https://wiki.oroboros.at/api.php?action=feedcontributions&user=Harrison+DK&feedformat=atomBioblast - User contributions [en]2024-03-29T00:43:42ZUser contributionsMediaWiki 1.36.1https://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90947Talk:Safranin2015-06-02T14:18:06Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods that may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, a simple two point calibration for safranin concentration is recommended as a minimum. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. <br />
<br />
If quantification is required, a multiple point calibration could be used to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when should the safranin be introduced into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the markers. However, the very different absorption properties of the solution without the sample compared to that with the sample means that sensitivity will probably be quite different. When the safranin is injected after the sample, the sensitivity will be correct but safranin is immediately taken up by the sample. To a certain degree this can be compensated for by placing a short mark immediately after the injection but the accuracy of this compensation will depend on the speed of uptake of safranin by the sample. However, If full quantification is not required, any small error here should be negligible. If the display of fluorescence intensity in a plot together with those from two different sensors is desired, displaying roughly calibrated safranin concentrations could be a recommended approach, otherwise there will be an offset between traces from the two other sensors. Indeed, just for the purpose of normalization between different sensors, even calibration without biological material would be suitable. The y axis of the resulting plot could be labelled "arbitrary units" - a facility possible in newer releases of [[DatLab]]. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Insufficient data is available to define "safe" safranin concentrations for different sample types. Please add your own experiences here. [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The chemical used should be tested for this effect in a background run without a biological sample. If necessary corrections should be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect. A significant effect has also been found with ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90946Talk:Safranin2015-06-02T14:10:37Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods that may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, a simple two point calibration for safranin concentration is recommended as a minimum. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. <br />
<br />
If quantification is required, a multiple point calibration could be used to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when should the safranin be introduced into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the markers. However, the very different absorption properties of the solution without the sample compared to that with the sample means that sensitivity will probably be quite different. When the safranin is injected after the sample, the sensitivity will be correct but safranin is immediately taken up by the sample. To a certain degree this can be compensated for by placing a short mark immediately after the injection but the accuracy of this compensation will depend on the speed of uptake of safranin by the sample. However, If full quantification is not required, any small error here should be negligible. If the display of fluorescence intensity in a plot together with plots from two different sensors is desired, this (displaying roughly calibrated safranin concentrations) could be a recommended approach, otherwise there will be an offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be suitable, maybe labelling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Insufficient data is available to define "safe" safranin concentrations for different sample types. Please add your own experiences here. [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The chemical used should be tested for this effect in a background run without a biological sample. If necessary corrections should be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect. A significant effect has also been found with ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90945Talk:Safranin2015-06-02T14:06:01Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods that may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, as a minimum, a simply two point calibration for safranin concentration is recommended. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. <br />
<br />
If quantification is required, a multiple point calibration could be used to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when should the safranin be introduced into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the markers. However, the very different absorption properties of the solution without the sample compared to that with the sample means that sensitivity will probably be quite different. When the safranin is injected after the sample, the sensitivity will be correct but safranin is immediately taken up by the sample. To a certain degree this can be compensated for by placing a short mark immediately after the injection but the accuracy of this compensation will depend on the speed of uptake of safranin by the sample. However, If full quantification is not required, any small error here should be negligible. If the display of fluorescence intensity in a plot together with plots from two different sensors is desired, this (displaying roughly calibrated safranin concentrations) could be a recommended approach, otherwise there will be an offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be suitable, maybe labelling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Insufficient data is available to define "safe" safranin concentrations for different sample types. Please add your own experiences here. [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The chemical used should be tested for this effect in a background run without a biological sample. If necessary corrections should be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect. A significant effect has also been found with ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90944Talk:Safranin2015-06-02T14:02:24Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, as a minimum, a simply two point calibration for safranin concentration is recommended. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. <br />
<br />
If quantification is required, a multiple point calibration could be used to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when should the safranin be introduced into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the markers. However, the very different absorption properties of the solution without the sample compared to that with the sample means that sensitivity will probably be quite different. When the safranin is injected after the sample, the sensitivity will be correct but safranin is immediately taken up by the sample. To a certain degree this can be compensated for by placing a short mark immediately after the injection but the accuracy of this compensation will depend on the speed of uptake of safranin by the sample. However, If full quantification is not required, any small error here should be negligible. If the display of fluorescence intensity in a plot together with plots from two different sensors is desired, this (displaying roughly calibrated safranin concentrations) could be a recommended approach, otherwise there will be an offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be suitable, maybe labelling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Insufficient data is available to define "safe" safranin concentrations for different sample types. Please add your own experiences here. [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The chemical used should be tested for this effect in a background run without a biological sample. If necessary corrections should be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect. A significant effect has also been found with ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90943Talk:Safranin2015-06-02T13:57:01Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, as a minimum, a simply two point calibration for safranin concentration is recommended. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. <br />
<br />
If quantification is required, a multiple point calibration could be used to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when should the safranin be introduced into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the markers. However, the very different absorption properties of the solution without the sample compared to that with the sample means that sensitivity will probably be quite different. When the safranin is injected after the sample, the sensitivity will be correct but safranin is immediately taken up by the sample. To a certain degree this can be compensated for by placing a short mark immediately after the injection but the accuracy of this compensation will depend on the speed of uptake of safranin by the sample. However, If full quantification is not required, any small error here should be negligible. If the display of fluorescence intensity in a plot together with plots from two different sensors is desired, this (displaying roughly calibrated safranin concentrations) could be a recommended approach, otherwise there will be an offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be suitable, maybe labelling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90927Talk:Safranin2015-06-02T13:39:14Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, as a minimum, a simply two point calibration for safranin concentration is recommended. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. <br />
<br />
If you want to go into quantification you could use a multiple point calibration to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is anyway based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when to introduce the safranin into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the marks but due to totally different absorption properties of the solution without sample the sensitivity will probable be different than with the sample in the chamber. When you inject the safranin after the sample , the sensitivity will be correct but you have to be aware that safranin is immediately taken up by the sample. To a certain degree this can be taken into account by placing a short mark immediately after the injection. I am not yet certain if the safranin uptake is slow enough for this to be a precise solution. If your are not going for quantification any small error here should not be a problem. If you just want to display fluorescence intensity in a plot but have to compare results from two different sensors, this (displaying roughly calibrated safranin concentrations) could be a good approach, otherwise you will have a offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be fine, maybe calling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90915Talk:Safranin2015-06-02T13:20:15Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, referred to here as the [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and in addition to the SUIT protocol.<br />
The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:<br />
<br />
# Deciding on a safranin concentration and sample-to-safranin concentration ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.<br />
<br />
<br />
If using the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether and if yes what kind of quantification you want to do. You will have to remind me, are you working with isolated mitochondria or with anything else? I personally see safranin mainly as a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature is definitely non linear. As I first step please have a look at the literature (people using safranin in cuvettes) and see what calibration procedures (and when they did introduce the safranin !) they used. These scientist have certainly more experience with this then we. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], you will find no calibration was done and only the plot with the fluorescence intensities in presented. Actually, I think this a quite honest approach. Non the less, independent of your final format for publication, as a minimum I would do a simply 2 point calibration for safranin concentration. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. If you want to go into quantification you could use a multiple point calibration to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is anyway based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when to introduce the safranin into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the marks but due to totally different absorption properties of the solution without sample the sensitivity will probable be different than with the sample in the chamber. When you inject the safranin after the sample , the sensitivity will be correct but you have to be aware that safranin is immediately taken up by the sample. To a certain degree this can be taken into account by placing a short mark immediately after the injection. I am not yet certain if the safranin uptake is slow enough for this to be a precise solution. If your are not going for quantification any small error here should not be a problem. If you just want to display fluorescence intensity in a plot but have to compare results from two different sensors, this (displaying roughly calibrated safranin concentrations) could be a good approach, otherwise you will have a offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be fine, maybe calling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90913Talk:Safranin2015-06-02T13:07:59Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Summarising, the differences between the safranin and TPP methods are:<br />
<br />
* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.<br />
* If quantification of differences is sufficient then safranin and TPP are both suitable.<br />
* The type of sample may make one method or the other more suitable.<br />
* The decision also relies on the types of calibration methods may be required.<br />
* There is probably always a benefit of having both methods available.<br />
* [[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
'''More input from users to this important topic would be welcome. Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, lets call it [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, lets call it "specific protocol". Unlike other methods neither safranin nor TPP require large modifications of the "SUIT protocol",beside excluding incompatible chemicals. That is both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and additional to the SUIT protocol.<br />
The "specific protocol" for safranin is rather limited. In the (till now) usual case of doing it in a cuvette of a spectrofluorometer this would include:<br />
<br />
# Deciding on a safranin concentration and sample to safranin ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by an other method is not done in the same experiment.<br />
<br />
<br />
For the use with the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether and if yes what kind of quantification you want to do. You will have to remind me, are you working with isolated mitochondria or with anything else? I personally see safranin mainly as a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature is definitely non linear. As I first step please have a look at the literature (people using safranin in cuvettes) and see what calibration procedures (and when they did introduce the safranin !) they used. These scientist have certainly more experience with this then we. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], you will find no calibration was done and only the plot with the fluorescence intensities in presented. Actually, I think this a quite honest approach. Non the less, independent of your final format for publication, as a minimum I would do a simply 2 point calibration for safranin concentration. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. If you want to go into quantification you could use a multiple point calibration to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is anyway based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when to introduce the safranin into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the marks but due to totally different absorption properties of the solution without sample the sensitivity will probable be different than with the sample in the chamber. When you inject the safranin after the sample , the sensitivity will be correct but you have to be aware that safranin is immediately taken up by the sample. To a certain degree this can be taken into account by placing a short mark immediately after the injection. I am not yet certain if the safranin uptake is slow enough for this to be a precise solution. If your are not going for quantification any small error here should not be a problem. If you just want to display fluorescence intensity in a plot but have to compare results from two different sensors, this (displaying roughly calibrated safranin concentrations) could be a good approach, otherwise you will have a offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be fine, maybe calling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90910Talk:Safranin2015-06-02T13:00:30Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers, who had already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry-over of inhibitors by the electrode, etc.). Safranin can be added to many standard protocols and the changes in fluorescence intensity (at least qualitatively) can be followed in parallel to the respiration measurements without going to all the extra steps necessary for TPP measurements.<br />
<br />
Therefore important considerations re safranin / TPP are:<br />
<br />
* Are absolute values required?<br />
* Is at least a quantification of differences (absolute differences) required?<br />
* What types of sample are of interest?<br />
* Which calibration methods are available?<br />
* There is probably always a benefit of having both methods available.<br />
<br />
[[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
<br />
'''More input from users to this important topic is required! Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, lets call it [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, lets call it "specific protocol". Unlike other methods neither safranin nor TPP require large modifications of the "SUIT protocol",beside excluding incompatible chemicals. That is both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and additional to the SUIT protocol.<br />
The "specific protocol" for safranin is rather limited. In the (till now) usual case of doing it in a cuvette of a spectrofluorometer this would include:<br />
<br />
# Deciding on a safranin concentration and sample to safranin ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by an other method is not done in the same experiment.<br />
<br />
<br />
For the use with the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether and if yes what kind of quantification you want to do. You will have to remind me, are you working with isolated mitochondria or with anything else? I personally see safranin mainly as a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature is definitely non linear. As I first step please have a look at the literature (people using safranin in cuvettes) and see what calibration procedures (and when they did introduce the safranin !) they used. These scientist have certainly more experience with this then we. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], you will find no calibration was done and only the plot with the fluorescence intensities in presented. Actually, I think this a quite honest approach. Non the less, independent of your final format for publication, as a minimum I would do a simply 2 point calibration for safranin concentration. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. If you want to go into quantification you could use a multiple point calibration to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is anyway based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when to introduce the safranin into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the marks but due to totally different absorption properties of the solution without sample the sensitivity will probable be different than with the sample in the chamber. When you inject the safranin after the sample , the sensitivity will be correct but you have to be aware that safranin is immediately taken up by the sample. To a certain degree this can be taken into account by placing a short mark immediately after the injection. I am not yet certain if the safranin uptake is slow enough for this to be a precise solution. If your are not going for quantification any small error here should not be a problem. If you just want to display fluorescence intensity in a plot but have to compare results from two different sensors, this (displaying roughly calibrated safranin concentrations) could be a good approach, otherwise you will have a offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be fine, maybe calling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90897Talk:Safranin2015-06-02T12:52:47Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally differently. Unlike the TPP method the relationship between fluorescence and membrane potential is entirely empirical. A linear relationship between fluorescence intensity and mitochondrial membrane potential is found for certain ranges and ratios of safranin and mitochondrial concentrations. In order to obtain quantitative values for mitochondrial membrane potential from the safranin method two caveats apply:<br />
* It must be established that the relationship is linear (or at least known) for the experimental conditions.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers who had the safranin method established (with a spectrofluormeter) to obtain also a TPP electrode.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry over of inhibitors by the electrode, ...). Safranin can be added to many standard protocols and the changes (at least qualitatively ) can be followed in parallel to the respiration measurement without going to all the extra steps necessary for TPP measurements.<br />
<br />
Therefore important considerations re safranin / TPP are:<br />
<br />
* Are absolute values required?<br />
* Is at least a quantification of differences (absolute differences) required?<br />
* What types of sample are of interest?<br />
* Which calibration methods are available?<br />
* There is probably always a benefit of having both methods available.<br />
<br />
[[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
<br />
'''More input from users to this important topic is required! Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, lets call it [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, lets call it "specific protocol". Unlike other methods neither safranin nor TPP require large modifications of the "SUIT protocol",beside excluding incompatible chemicals. That is both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and additional to the SUIT protocol.<br />
The "specific protocol" for safranin is rather limited. In the (till now) usual case of doing it in a cuvette of a spectrofluorometer this would include:<br />
<br />
# Deciding on a safranin concentration and sample to safranin ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by an other method is not done in the same experiment.<br />
<br />
<br />
For the use with the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether and if yes what kind of quantification you want to do. You will have to remind me, are you working with isolated mitochondria or with anything else? I personally see safranin mainly as a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature is definitely non linear. As I first step please have a look at the literature (people using safranin in cuvettes) and see what calibration procedures (and when they did introduce the safranin !) they used. These scientist have certainly more experience with this then we. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], you will find no calibration was done and only the plot with the fluorescence intensities in presented. Actually, I think this a quite honest approach. Non the less, independent of your final format for publication, as a minimum I would do a simply 2 point calibration for safranin concentration. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. If you want to go into quantification you could use a multiple point calibration to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is anyway based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when to introduce the safranin into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the marks but due to totally different absorption properties of the solution without sample the sensitivity will probable be different than with the sample in the chamber. When you inject the safranin after the sample , the sensitivity will be correct but you have to be aware that safranin is immediately taken up by the sample. To a certain degree this can be taken into account by placing a short mark immediately after the injection. I am not yet certain if the safranin uptake is slow enough for this to be a precise solution. If your are not going for quantification any small error here should not be a problem. If you just want to display fluorescence intensity in a plot but have to compare results from two different sensors, this (displaying roughly calibrated safranin concentrations) could be a good approach, otherwise you will have a offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be fine, maybe calling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Safranin&diff=90893Talk:Safranin2015-06-02T12:43:54Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== TPP vs safranin ==<br />
'''Question:'''<br />
I guess I would also like to know you guys opinion on TPP vs. safranin for measuring membrane potential. Which is better? Which is more reliable? Which is easier?<br />
<br />
<br />
'''Answer by OROBOROS:'''<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see<br />
<br />
[[Mitochondrial membrane potential]]<br />
and especially<br />
<br />
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]<br />
<br />
Absolute measurements of membrane potential in isolated mitochondria can be obtained with the TPP method by using unspecific binding correction factors that have been obtained via a "calibration" against the Rb radio labeling method. For other sample types "absolute" quantification has still to be developed, see the link above.<br />
<br />
The safranin method works totally different. Unlike the TPP method the relationship between fluorescence and membrane potential is totally empirical only. For certain safranin and mitochondrial concentrations and for certain ratios of these two parameters, a linear relationship between fluorescence intensity and mitochondrial membrane potential was found. To get quantitative values for mitochondrial membrane potential from the safranin method two conditions must be fulfilled:<br />
* It must be established that the relationship is linear (or at least known) for the experimental condition.<br />
* Some calibration is necessary, even for an established linear relationship.<br />
<br />
One method to calibrate the safranin method is actually to use the TPP method, though you will find other methods in the literature. That in fact was the driving force for some customers who had the safranin method established (with a spectrofluormeter) to obtain also a TPP electrode.<br />
<br />
On the the other hand the safranin method is far easier to handle in the lab than the TPP method (no extra electrodes, no problem with carry over of inhibitors by the electrode, ...). Safranin can be added to many standard protocols and the changes (at least qualitatively ) can be followed in parallel to the respiration measurement without going to all the extra steps necessary for TPP measurements.<br />
<br />
Therefore important considerations re safranin / TPP are:<br />
<br />
* Are absolute values required?<br />
* Is at least a quantification of differences (absolute differences) required?<br />
* What types of sample are of interest?<br />
* Which calibration methods are available?<br />
* There is probably always a benefit of having both methods available.<br />
<br />
[[Talk:Safranin#Inhibition_by_safranin|Inhibition by safranin]] may also be a major disadvantage of the safranin method.<br />
<br />
<br />
<br />
'''More input from users to this important topic is required! Please contact instruments@oroboros.at to get an account for this wiki to contribute to this discussion'''!<br />
<br />
'''Follow up question''': Whats the difference between a TPP and a safranin protocol?<br />
<br />
'''Answer:''' We have to discern between the protocol for the addition of substrates, uncouplers, etc, lets call it [[Substrate-uncoupler-inhibitor titration|"SUIT protocol"]] and specific additional steps necessary for safranin or TPP, lets call it "specific protocol". Unlike other methods neither safranin nor TPP require large modifications of the "SUIT protocol",beside excluding incompatible chemicals. That is both methods can be used with a wide range of different SUIT protocols. The specific protocol for TPP comprises calibration of the TPP electrode, introduction of sample into the chamber containing TPP, etc. All this is done "around" and additional to the SUIT protocol.<br />
The "specific protocol" for safranin is rather limited. In the (till now) usual case of doing it in a cuvette of a spectrofluorometer this would include:<br />
<br />
# Deciding on a safranin concentration and sample to safranin ratio.<br />
# Selection of excitation and emission wavelength and bandwidth.<br />
# Addition of safranin.<br />
# Addition of sample and start of SUIT protocol.<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
<br />
Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by an other method is not done in the same experiment.<br />
<br />
<br />
For the use with the [[O2k-Fluorescence LED2-Module]] the second step is replaced by selecting an appropriate sensor, filter set and light intensity, see [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]]. <br />
<br />
For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].<br />
<br />
<br />
<br />
--[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)<br />
<br />
== Safranin calibration ==<br />
<br />
'''Question:''' <br />
We've been using the O2K unit to measure ROS production using amplex ultra red, and so far I think it has been going well! <br />
I now would like to use saffranin to measure membrane potential. I just read up on some info on the O2K website. Do I have to calibrate the signal as I do with amplex red? And if so, what should I use? Is it done in the same way (with the same excel spreadsheet?<br />
<br />
'''Answer:'''<br />
<br />
'''Update:''' please see also: [[MiPNet19.19 Safranin Data Acquisition and Analysis]] and [[Krumschnabel 2014 Methods Enzymol]].<br />
That depends on whether and if yes what kind of quantification you want to do. You will have to remind me, are you working with isolated mitochondria or with anything else? I personally see safranin mainly as a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample ratios plus well defined absolute safranin concentrations there is linear correlation between detected safranin fluorescence and mitochondrial membrane potential, e.g. [[Figueira 2012 Methods Mol Biol]].<br />
<br />
For other samples (e.g. permeabilised cells) this relation, as shown in the literature is definitely non linear. As I first step please have a look at the literature (people using safranin in cuvettes) and see what calibration procedures (and when they did introduce the safranin !) they used. These scientist have certainly more experience with this then we. However, in many papers, e.g. [[Komary 2010 Biochim Biophys Acta]], you will find no calibration was done and only the plot with the fluorescence intensities in presented. Actually, I think this a quite honest approach. Non the less, independent of your final format for publication, as a minimum I would do a simply 2 point calibration for safranin concentration. This you can do directly in DatLab in the calibration window. This should be enough if you mainly want to look on the data in a qualitative way but will still allow you to compare results from different sensors and do a correction for any chemical background effects. If you want to go into quantification you could use a multiple point calibration to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is anyway based on the safranin fluorescence signal and not the actual safranin concentration. If you want to do a multi point calibration you can indeed use the template for Amplex red. So when to introduce the safranin into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the marks but due to totally different absorption properties of the solution without sample the sensitivity will probable be different than with the sample in the chamber. When you inject the safranin after the sample , the sensitivity will be correct but you have to be aware that safranin is immediately taken up by the sample. To a certain degree this can be taken into account by placing a short mark immediately after the injection. I am not yet certain if the safranin uptake is slow enough for this to be a precise solution. If your are not going for quantification any small error here should not be a problem. If you just want to display fluorescence intensity in a plot but have to compare results from two different sensors, this (displaying roughly calibrated safranin concentrations) could be a good approach, otherwise you will have a offset between traces from the two different sensors. Indeed, just for the purpose of normalization between different sensors even calibration without biological material would be fine, maybe calling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of [[DatLab]] 5. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)<br />
<br />
== Inhibition by safranin ==<br />
<br />
Safranin inhibits mitochondrial function ([[Krumschnabel 2014 Methods Enzymol]]). Always check the influence of the safranin concentration used on the respiratory rate. Not enough data is available to define "safe" safranin concentrations for different sample types. Please add your experiences here! [[User:Fasching Mario|Fasching Mario]] 09:12, 10 January 2013 (CET)<br />
<br />
This problem seems to be quite severe. [[User:Laner Verena|Laner Verena]] 14:36, 16 April 2013 (CEST)<br />
<br />
== Safranin chemical background ==<br />
<br />
Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The used chemical should be tested for this effect in a background run without biological sample. If necessary corrections have to be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect, however, we also found a significant effect for ADP. [[User:Fasching Mario|Fasching Mario]] 09:19, 10 January 2013 (CET)<br />
<br />
<br />
'''Substances with an effect on the fluorescence signal of safranin'''<br />
* ADP (D)<br />
* Cytochrome ''c'' (c)<br />
* Succinate (S)<br />
* Rotenone (Rot)<br />
* Ascorbate (As)<br />
* TMPD (Tm)<br />
<br />
<br />
'''Substances without an effect on the fluorescence signal of safranin'''<br />
<br />
* Pyruvate (P)<br />
* Malate (M)<br />
* Glutamate (G)<br />
* Digitonin (Dig)<br />
* Oligomycin (Omy)<br />
* FCCP (U)<br />
* Malonic acid (Mna)<br />
* Antimycin A (Ama)<br />
* DMSO <br />
* Ethanol<br />
* H<sub>2</sub>O<sub>2</sub><br />
* H<sub>2</sub>O</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:O2k-Open_Support&diff=90186Talk:O2k-Open Support2015-05-24T15:27:46Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== Technical service - pages in preparation ==<br />
* Introduction to O2k-technical support: [[Media:Technical Support.pdf|'''»O2k-technical support.pdf''']]<br />
* The pages are listed in the [http://wiki.oroboros.at/index.php/Category:Technical_service Browse data page], starting with "Talk:.."<br />
* http://wiki.oroboros.at/index.php/Special:WhatLinksHere/POS_calibration<br />
* [[MiPNet06.03 POS-Calibration-SOP]] - OroboPOS technical support: many repeats, text lacks style. Reference to [[MiPNet12.08]]???<br />
* [[MiPNet14.06 InstrumentalBackground]] - Flux at closed chamber near to air saturation<br />
<br />
<br />
== Technical service by telephone ==<br />
We prefer to give technical assistance („trouble shooting“) via Email. We are aware that, especially if there is an urgent problem, some end-users would prefer to communicate by telephone. However, we are convinced that there are very good reasons for our approach:<br />
<br />
Communication by Email <br />
* gives us the time to thoroughly read the customers problem description, analyze in detail the files sent to us and THINK about our proposals BEFORE suggesting them to you, <br />
* gives the customer time to read our suggestions carefully and to read any additional material (websites , manual !) that we proposed,<br />
* gives the customer time to properly collect (and document) the data that we asked her/him to send, <br />
* allows our technical service to be operated "by scientists, for scientists". The members of our technical service team work on their own project, keeping up to date with all technical development. However, this means they will not be available for phone conversation while running their experiments.<br />
For a few special cases telephone conversations may actually make sense. In this case we will of course gladly arrange for such a phone call (planned beforehand).<br />
<br />
When phoning Oroboros Instruments you will be connected to our administrative team. They will be glad to help you in administrative questions and point you to the right starting places for trouble shooting. Please understand that it will not be helpful to describe to them any technical details related to a problem on the phone. Even our technical staff prefer to get this information by e-mail, information relayed via non technical experts would be even less helpful. Therefore, this information should be sent by e-mail and include as much of the information gathered during trouble shooting (see the main page) as possible, including a DatLab file (not a screen shot) of a [[Sensor test]].<br />
<br />
[[User:Fasching Mario|Fasching Mario]] 09:35, 17 April 2013 (CEST)<br />
<br />
[[Image:BB-Bioblast.jpg|left|30px|link=Bioblast:About|Bioblast wiki]]<br />
<br />
== Popular Bioblast page ==<br />
[[O2k-technical support and open innovation]] has been accessed more than <br />
:* 15,000 times (2014-08-28)<br />
:* 10,000 times (2014-04-17)<br />
:* 5,000 times (2012-06-12)</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:O2k-Open_Support&diff=90185Talk:O2k-Open Support2015-05-24T15:22:32Z<p>Harrison DK: </p>
<hr />
<div>__TOC__<br />
<br />
== Technical service - pages in preparation ==<br />
* Introduction to O2k-technical support: [[Media:Technical Support.pdf|'''»O2k-technical support.pdf''']]<br />
* The pages are listed in the [http://wiki.oroboros.at/index.php/Category:Technical_service Browse data page], starting with "Talk:.."<br />
* http://wiki.oroboros.at/index.php/Special:WhatLinksHere/POS_calibration<br />
* [[MiPNet06.03 POS-Calibration-SOP]] - OroboPOS technical support: many repeats, text lacks style. Reference to [[MiPNet12.08]]???<br />
* [[MiPNet14.06 InstrumentalBackground]] - Flux at closed chamber near to air saturation<br />
<br />
<br />
== Technical service by telephone ==<br />
We prefer to give technical assistance („trouble shooting“) per Email. We are aware that, especially if there is an urgent problem, some end-users would prefer to communicate by telephone. However, we are convinced that there are very good reasons for our approach:<br />
<br />
Communication by Email <br />
* gives us the time to thoroughly read the customers problem description, analyze in detail the files sent to us and THINK about our proposals BEFORE suggesting them to you, <br />
* gives the customer time to read our suggestions carefully and to read any additional material (websites , manual !) that we proposed,<br />
* gives the customer time to properly collect (and document) the data that we asked her/him to send, <br />
* allows our technical service to be operated "by scientists, for scientists". The members of our technical service team work on their own project, keeping up to date with all technical development. However, this means they will not be available for phone conversation while running their experiments.<br />
For a few special cases telephone conversations may actually make sense. In this case we will of course gladly arrange for such a phone call (planned beforehand).<br />
<br />
When phoning Oroboros Instruments you will be connected to our administrative team. They will be glad to help you in administrative questions and point you to the right starting places for trouble shooting. Please understand that it will not be helpful to describe to them any technical details related to a problem on the phone. Even our technical staff prefers to get this information by e-mail, information relayed via non technical experts would be even less helpful. Therefore, this information should be sent by e-mail and include as much of the information gathered during trouble shooting (see the main page) as possible, including a DatLab file (not a screen shot) of a [[Sensor test]].<br />
<br />
[[User:Fasching Mario|Fasching Mario]] 09:35, 17 April 2013 (CEST)<br />
<br />
[[Image:BB-Bioblast.jpg|left|30px|link=Bioblast:About|Bioblast wiki]]<br />
<br />
== Popular Bioblast page ==<br />
[[O2k-technical support and open innovation]] has been accessed more than <br />
:* 15,000 times (2014-08-28)<br />
:* 10,000 times (2014-04-17)<br />
:* 5,000 times (2012-06-12)</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Setting_the_oxygen_concentration&diff=90181Talk:Setting the oxygen concentration2015-05-23T16:30:23Z<p>Harrison DK: </p>
<hr />
<div>{{Technical service}}<br />
Question: Can we set the oxygen concentration in the Oroboros-2k to desired levels?<br />
<br />
Answer:<br />
Yes.<br />
<br />
It is always possible to increase the oxygen concentration using the combination of catalase in the medium and injections of H2O2, as described in [[MiPNet14.13 Medium-MiR06| MiPNet14.3]]. By using the [[TIP2k]] the oxygen concentration can be maintained between well defined limits, either using H2O2 or (for very low oxygen concentrations) air saturated medium in the TIP syringes. We call this an "oxystat" approach and supply appropriate templates for controlling the TIP2k. See also [http://www.oroboros.at/index.php?id=o2k-tip2k-manual MiPNet12.10]. <br />
<br />
This leaves the question of how low oxygen levels can be reached in the first place: In a biological experiment this may be done by inserting a N2 bubble into the chamber, waiting until the desired level of oxygen is reached and then destroying the air bubble by closing the stoppers. However, most often the desired starting value is simply reached by waiting until the sample has consumed the required amount of oxygen and then starting the "oxystat".<br />
For calibration and instrumental oxygen background purposes we use Dithionite to reduce oxygen levels, however this is not recommended in the presence of biological samples.<br />
<br />
{{#set:Technical service=Chamber|Technical service=O2 signal}}<br />
<br />
<br> <br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Setting_the_oxygen_concentration&diff=90180Talk:Setting the oxygen concentration2015-05-23T16:28:29Z<p>Harrison DK: </p>
<hr />
<div>{{Technical service}}<br />
Question: Can we set the oxygen concentration in the Oroboros-2k to desired levels?<br />
<br />
Answer:<br />
Yes.<br />
<br />
It is always possible to increase the oxygen concentration using the combination of catalase in the medium and injections of H2O2, as described in [[MiPNet14.13 Medium-MiR06| MiPNet14.3]]. By using the [[TIP2k]] the oxygen concentration can be maintained between well defined limits, either using H2O2 or (for very low oxygen concentrations) air saturated medium in the TIP syringes. We call this an "oxystat" approach and supply appropriate templates for controlling the TIP2k in. See also [http://www.oroboros.at/index.php?id=o2k-tip2k-manual MiPNet12.10]. <br />
<br />
This leaves the question how low oxygen levels are reached in the first place: In a biological experiment this may be done by inserting a N2 bubble into the chamber, waiting until the desired level of oxygen is reached and then destroying the air bubble by closing the stoppers. However, most often the desired starting value is simply reached by waiting until the sample has consumed the required amount of oxygen and then starting the "oxystat".<br />
For calibration and instrumental oxygen background purposes we use Dithionite to reduce oxygen levels, however this is not recommended in the presence of biological samples.<br />
<br />
{{#set:Technical service=Chamber|Technical service=O2 signal}}<br />
<br />
<br> <br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:O2k-Peltier_Temperature_Control&diff=90179Talk:O2k-Peltier Temperature Control2015-05-23T16:15:21Z<p>Harrison DK: </p>
<hr />
<div>Previous Product ID 21020<br />
{{Technical support}}<br />
__TOC__<br />
<br />
== Very low temperatures ==<br />
<br />
'''Question:''' Is it possible to use the oxygraph with a chamber temperature of 0 °C?<br />
<br />
Our system is specified for a chamber temperature down to 4 °C at 25 °C room temperature and down to 2 °C at "lower ambient temperature", see<br />
[http://www.oroboros.at/index.php?id=o2k-specifications| specifications].<br />
Lower ambient temperature does not necessarily mean a "cool room". For a chamber temperature of 2 °C, an air conditioned room will usually suffice.<br />
While we can only guarantee the working of the instrument within these limits, it is possible to reach a chamber temperature of 0 °C at a "lower ambient temperature". In general, however, this may require a "cool room". For this purpose, the main unit itself can be exposed to ambient temperatures down to just above 0°C (all the electronic components are rated at least down to 0 °C, but 0 °C room temperature should be avoided due to condensation concerns). Also, for very low room temperatures above 0 °C, some means of humidity control should be used.<br />
We recently performed a short test and were able to reach 0 °C chamber temperature quite easily at a room temperature of about 14 °C. In order to maintain this temperature, the cooling power of the Peltier element reached 95% of maximum. Thus, a "cool room" would probably be necessary for longer experiments.<br />
<br />
'''Update for O2k Series E:'''<br />
Because Series E Oxygraphs have a more efficient temperature management system the experiment described above was repeated with a Series E O2k. In this case it was not necessary to reduce the room temperature. [[User:Fasching Mario|Fasching Mario]] 14:39, 4 February 2013 (CET) <br />
<br />
Details: O2K - 0°C block temp. - stable Peltier power was reached after about 1:05 h from setting block temp. from stable 37°C to 0°C. - stable Peltier power was about -83 %. - room temperature was 21.1°C [[User:Capek Ondrej|Capek Ondrej]] 14:36, 4 February 2013 (CET)<br />
<br />
<br />
<br />
{{#set:Technical service=Main unit}}<br />
<br />
__SHOWFACTBOX__<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:O2k-Peltier_Temperature_Control&diff=90178Talk:O2k-Peltier Temperature Control2015-05-23T16:01:41Z<p>Harrison DK: </p>
<hr />
<div>Previous Product ID 21020<br />
{{Technical support}}<br />
__TOC__<br />
<br />
== Very low temperatures ==<br />
<br />
'''Question:''' Is it possible to use the oxygraph with a chamber temperature of 0 °C?<br />
<br />
Our system is specified down to 4 °C at 25 °C room temperature and down to 2 °C at "lower ambient temperature", see<br />
[http://www.oroboros.at/index.php?id=o2k-specifications| specifications].<br />
Lower ambient temperature does not necessarily mean a "cool room". For 2 °C, an air conditioned room will usually suffice.<br />
While we can only guarantee the working of the instrument within these limits, it is possible to reach a chamber temperature of 0 °C at a "lower ambient temperature". In general, however, this may require a "cool room". For this purpose, the main unit itself can be exposed to temperatures down to just above 0°C (all the electronic components are rated at least down to 0 °C, but 0 °C room temperature should be avoided due to condensation concerns). Also, for very low room temperatures above 0 °C, humidity should be controlled by some means.<br />
We recently performed a short test and were able to reach 0 °C chamber temperature quite easily at a room temperature of about 14 °C. In order to maintain this temperature, the cooling power of the Peltier element reached 95% of maximum. Thus, some form of cool room would probably be necessary for longer experiments.<br />
<br />
'''Update for O2k Series E:'''<br />
Because Series E Oxygraphs have a more efficient temperature management system the experiment described above was repeated with a Series E O2k. In this case it was not necessary to reduce the room temperature. [[User:Fasching Mario|Fasching Mario]] 14:39, 4 February 2013 (CET) <br />
<br />
Details: O2K - 0°C block temp. - stable Peltier power was reached after about 1:05 h from setting block temp. from stable 37°C to 0°C. - stable Peltier power was about -83 %. - room temperature was 21.1°C [[User:Capek Ondrej|Capek Ondrej]] 14:36, 4 February 2013 (CET)<br />
<br />
<br />
<br />
{{#set:Technical service=Main unit}}<br />
<br />
__SHOWFACTBOX__<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:O2k-Peltier_Temperature_Control&diff=90177Talk:O2k-Peltier Temperature Control2015-05-23T15:59:57Z<p>Harrison DK: </p>
<hr />
<div>Previous Product ID 21020<br />
{{Technical support}}<br />
__TOC__<br />
<br />
== Very low temperatures ==<br />
<br />
'''Question:''' Is it possible to use the oxygraph with a chamber temperature of 0 °C?<br />
<br />
Our system is specified down to 4 °C at 25 °C room temperature and down to 2 °C at "lower ambient temperature", see<br />
[http://www.oroboros.at/index.php?id=o2k-specifications| specifications].<br />
Lower ambient temperature does not necessarily mean a "cool room". For 2 °C, an air conditioned room will usually suffice.<br />
While we can only guarantee the working of the instrument within these limits, it is possible to reach a chamber temperature of 0 °C at higher ambient temperatures. In general, however, this may require a "cool room". For this purpose, the main unit itself can be exposed to temperatures down to just above 0°C (all the electronic components are rated at least down to 0 °C, but 0 °C room temperature should be avoided due to condensation concerns). Also, for very low room temperatures above 0 °C, humidity should be controlled by some means.<br />
We recently performed a short test and were able to reach 0 °C chamber temperature quite easily at a room temperature of about 14 °C. In order to maintain this temperature, the cooling power of the Peltier element reached 95% of maximum. Thus, some form of cool room would probably be necessary for longer experiments.<br />
<br />
'''Update for O2k Series E:'''<br />
Because Series E Oxygraphs have a more efficient temperature management system the experiment described above was repeated with a Series E O2k. In this case it was not necessary to reduce the room temperature. [[User:Fasching Mario|Fasching Mario]] 14:39, 4 February 2013 (CET) <br />
<br />
Details: O2K - 0°C block temp. - stable Peltier power was reached after about 1:05 h from setting block temp. from stable 37°C to 0°C. - stable Peltier power was about -83 %. - room temperature was 21.1°C [[User:Capek Ondrej|Capek Ondrej]] 14:36, 4 February 2013 (CET)<br />
<br />
<br />
<br />
{{#set:Technical service=Main unit}}<br />
<br />
__SHOWFACTBOX__<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:O2k-Peltier_Temperature_Control&diff=90176Talk:O2k-Peltier Temperature Control2015-05-23T15:52:08Z<p>Harrison DK: </p>
<hr />
<div>Previous Product ID 21020<br />
{{Technical support}}<br />
__TOC__<br />
<br />
Previous Product ID 21020<br />
{{Technical service}}<br />
__TOC__<br />
<br />
== Very low temperatures ==<br />
<br />
'''Question:''' Is it possible to use the oxygraph with a chamber temperature of 0 °C?<br />
<br />
Our system is specified down to 4 °C at 25 °C room temperature and down to 2 °C at "lower ambient temperature", see<br />
[http://www.oroboros.at/index.php?id=o2k-specifications| specifications].<br />
Lower ambient temperature does not necessarily mean a "cool room". For 2 °C, an air conditioned room will usually suffice.<br />
While we can only guarantee the working of the instrument within these limits, it has been shown to be possible to go successfully to 0 °C. In general, this may require a "cool room". For this purpose, the main unit itself can be exposed to temperatures down to just above 0°C (all the electronic components are rated at least down to 0 °C but 0 °C room temperature should be avoided due to condensation concerns). Also, for very low room temperatures above 0 °C, humidity should be controlled by some means.<br />
We recently performed a short test and were able to reach 0 °C chamber temperature quite easily at a room temperature of about 14 °C. In order to maintain this temperature, the cooling power of the Peltier element reached 95% of maximum. Thus, some form of cool room would probably be necessary for longer experiments.<br />
<br />
'''Update for O2k Series E:'''<br />
Because Series E Oxygraphs have a more efficient temperature management system the experiment described above was repeated with a Series E O2k. In this case it was not necessary to reduce the room temperature. [[User:Fasching Mario|Fasching Mario]] 14:39, 4 February 2013 (CET) <br />
<br />
Details: O2K - 0°C block temp. - stable Peltier power was reached after about 1:05 h from setting block temp. from stable 37°C to 0°C. - stable Peltier power was about -83 %. - room temperature was 21.1°C [[User:Capek Ondrej|Capek Ondrej]] 14:36, 4 February 2013 (CET)<br />
<br />
<br />
<br />
{{#set:Technical service=Main unit}}<br />
<br />
__SHOWFACTBOX__<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Different_O2_fluxes_in_left_and_right_chamber&diff=90171Talk:Different O2 fluxes in left and right chamber2015-05-23T14:08:24Z<p>Harrison DK: </p>
<hr />
<div>{{Technical support}}<br />
<br />
'''Question:''' In our experiments we get consistently higher fluxes in chamber A as compared to chamber B.<br />
<br />
'''Answer:''' To exclude instrumental causes please follow the procedure described in the section <br />
'''Calibration and quality control of the OroboPOS (O2k-SOP)''' in [[MiPNet06.03 POS-Calibration-SOP]].<br />
<br />
Possible instrumental and protocol related causes for fluxes differences between chambers:<br />
<br />
* Incorrect O2 calibration in one chamber, see [[MiPNet06.03 POS-Calibration-SOP]].<br />
* Incorrect chamber volume calibration: The total amount of oxygen in the chamber with the smaller volume will be smaller. Therefore, the O2 concentration will drop faster resulting in a higher flux, see [[MiPNet19.18A O2k-Start]].<br />
* ##Incorrect background parameters: incorrect background parameters will cause an O2 concentration-dependent offset to the flux, the value of the offset will be independent of the absolute value of the flux. E.g. a constant offset of 10 pmol / s ml at ca 100 µM O2 concentration and 20 pmol/s ml at 300 µM O2 concentration could be caused by wrong background parameters, but a constant difference of e.g. 20% at very different absolute values of the flux can not be caused by wrong background parameters. In any case, it is advisable to check the background parameters (perform an instrumental O2 background experiment). See [[MiPNet14.06 InstrumentalBackground]].##<br />
* ##Sample injection: Scenario: One filling of a syringe is used to inject the sample into several chambers. Sedimentation starts to occur immediately and more sample will be injected into the first chamber than in any subsequent chambers. A remedy we apply is to fill the syringe with sample sufficient for 3 chambers and inject the first aliquot back to the sample stock solution before quickly injecting the rest into the chambers. Independently of this we recommend randomizing the assignment of the chambers to prevent systematic errors.##<br />
* Hydrophobic inhibitors, see [[MiPNet19.03 O2k-cleaning and ISS]].<br />
* Biological contamination, see [[Biological contamination]].<br />
<br />
Hardware defects: As hardware defects (sensors, electronics, etc.) have many negative effects (noisy signal, slow response, no signal, etc.) on experiments, it is difficult to see how a real hardware defect can cause a systematic error in flux calculation once a correct calibration of the O2 sensor (at air saturation and at zero oxygen) has been obtained. [[User:Fasching Mario|Fasching Mario]] 09:49, 7 November 2014 (CET)<br />
<br />
<br />
<br />
{{#set: Technical service =Chamber}}<br />
{{#set: Technical service =O2 signal}}<br />
<br />
[[category:Technical service]]<br />
<br />
<br />
<br />
<br />
__SHOWFACTBOX__</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Biological_contamination&diff=90170Talk:Biological contamination2015-05-23T13:54:52Z<p>Harrison DK: </p>
<hr />
<div>{{Technical support}}<br />
<br />
<br />
Biological contamination is caused by the growth of biological material (originating from samples or otherwise). It should not be confused with other kinds of chamber contamination mentioned in [[Cleaning the glass chamber]].<br />
<br />
Biological contamination of the chamber is typically characterized by a high O2 flux with a closed chamber near air saturation. It is detected by a [[sensor test]], an [[instrumental background test]] or by observing the flux at closed chamber for a short time routinely before any experiment. In the latter two cases the high flux contamination may be caused by biological contamination of the chamber itself or by a contaminated medium. This can be tested by observing the flux in pure water.<br />
<br />
The ideal remedial agent is 70% ethanol with 30% water (not 100% ethanol). Make sure the 70% ethanol does not contain any additives. E.g. 70% ethanol used in hospital settings may contain antiseptics (information provided by [[Garcia-Roves PM]]).<br />
Prevention of biological contamination of the chamber is primarily by storage under 70% ethanol. If repeated washing with 70% Ethanol does not remove an existing biological contamination, the glass chamber has to be cleaned as described for protein contamination under [[Cleaning the glass chamber]] by removing the glass chamber from the instrument and using a strong acid.<br />
<br />
<br />
{{#set: Technical service =Chamber}}<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Oxygen_flux_in_open_O2k-Chamber&diff=89643Talk:Oxygen flux in open O2k-Chamber2015-05-19T14:25:53Z<p>Harrison DK: </p>
<hr />
<div>__SHOWFACTBOX__<br />
<br />
<br />
<br />
= Mario Fasching =<br />
{{Technical service}}<br />
<br />
<br />
In an [[open chamber]] situation the liquid phase in the chamber is in equilibrium with the atmosphere. All oxygen consumed by the [[POS]] is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the non-background corrected oxygen flux (= first time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab) has to be zero. The background corrected flux is meaningless for the open chamber situation. This is because the background corrected flux corrects for consumption of oxygen by the sensor and diffusion into and out of the closed chamber. Since neither of the latter have any effect on the zero flux in the open chamber situation, an incorrect flux will result when background correction is applied. Only Layouts that display "O2 slope" uncorrected are suitable for assessing the O2 flux at open chamber. Such Layouts are: <br />
* 01 Calibration Exp Gr3-Temp<br />
* 02 Background Experiment<br />
* 04 Flux per Volume uncorrected<br />
* Z Trouble Shooting<br />
<br />
The observation of a zero flux with an open chamber is an important performance parameter. It indicates that thermal stability has been reached. Therefore no experiment should be started before a zero oxygen flux has been reached with an open chamber. The suggested criterion for "zero flux" is a flux between <br />
-1 pmol/(s ml) and + 1 pmol (s ml).<br />
<br />
<br />
'''Problem:''' <br />
* The uncorrected slope does not reach the interval +/- 1 pmol/(s ml) even after a prolonged time.<br />
<br />
'''Solutions:'''<br />
* The [[open chamber]] situation may not be ideal. Remove the stoppers completely and create a new gas phase as described in [[open chamber]].<br />
* The slope is always calculated from the calibrated O2 signal. Check the [[POS calibration]]<br />
* Observe the [[Raw signal]] and check if it is in the expected range<br />
* If the raw signal is unstable, and thereby creating an apparent O2 flux, follow the instructions for [[Locating a problem]].<br />
<br />
'''See also:'''<br />
<br />
POS calibration [http://www.oroboros.at/index.php?id=o2k-o2calibration MiPNet12.08]<br />
<br />
O2 Flux analysis [http://www.oroboros.at/index.php?id=o2k-datlab-fluxanalysis MiPNet12.09]<br />
<br />
{{#set:Technical service=O2 signal|Technical service=Chamber}}<br />
<br />
[[Category:Technical service]]<br />
<br />
__SHOWFACTBOX__</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:DatLab_plotting_of_O2_flux_per_mass%5C_unit_sample_or_flow_per_cell_don%27t_work&diff=89636Talk:DatLab plotting of O2 flux per mass\ unit sample or flow per cell don't work2015-05-19T14:24:07Z<p>Harrison DK: </p>
<hr />
<div>{{Technical support}}<br />
'''Problem:'''<br />
The plotting of the O2 flux per mass\unit sample or the flow per cell do not work - neither by selecting the plot manually in 'Graph - Select Plots' (in the menu) nor by choosing the appropriate layout from the menu 'Layout'.<br />
<br />
'''Cause:'''<br />
<br />
# Sample Concentration\Amount inside the 'Edit Experiment' window (menu 'Experiment - Edit') equal to 0<br />
# The information of the Sample Unit\Concentration\Amount inside the 'Edit Experiment' window (menu 'Experiment - Edit') hasn't been saved yet. Although the values inside this window are taken from the last experiment (measured with this DatLab installation, where the 'Save' button inside the 'Edit Experiment' has been pressed) and displayed at the beginning of a next experiments, they are not automatically saved.<br />
<br />
'''Solution:'''<br />
<br />
# Enter the Concentration\Amount different from 0<br />
# Inside the 'Edit Experiment' window (menu 'Experiment - Edit') review (or enter) the sample information (Unit\Concentration\Amount), click on the 'Save' button instead of 'Cancel' - then the appropriate plot can be selected (if the Concentration\Amount is not equal to 0).<br />
<br />
[[User:Capek Ondrej|Capek Ondrej]] 15:24, 2 April 2015 (CEST)<br />
<br />
<br />
{{#set:Technical service=DatLab}}<br />
<br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:DatLab_plotting_of_O2_flux_per_mass%5C_unit_sample_or_flow_per_cell_don%27t_work&diff=89629Talk:DatLab plotting of O2 flux per mass\ unit sample or flow per cell don't work2015-05-19T14:22:03Z<p>Harrison DK: </p>
<hr />
<div>{{Technical support}}<br />
'''Problem:'''<br />
The plotting of the O2 flux per mass\unit sample or the flow per cell do not work - neither by selecting the plot manually in 'Graph - Select Plots' (in the menu) nor by choosing the appropriate layout from the menu 'Layout'.<br />
<br />
'''Cause:'''<br />
<br />
# Sample Concentration\Amount inside the 'Edit Experiment' window (menu 'Experiment - Edit') equal to 0<br />
# The information of the Sample Unit\Concentration\Amount inside the 'Edit Experiment' window (menu 'Experiment - Edit') hasn't been saved yet. Although the values inside this window are taken from the last experiment (measured with this DatLab installation, where the 'Save' button inside the 'Edit Experiment' has been pressed) and displayed at the beginning of a next experiments, they are not automatically saved.<br />
<br />
'''Solution:'''<br />
<br />
# Enter the Concentration\Amount different from 0<br />
# Inside the 'Edit Experiment' window (menu 'Experiment - Edit') review (or enter) the sample information (Unit\Concentration\Amount), click on the 'Save' button instead of 'Cancel' - then the appropriate plot can be selected (if the Concentration\Amount is not equal to 0).<br />
<br />
[[User:Capek Ondrej|Capek Ondrej]] 15:24, 2 April 2015 (CEST)<br />
<br />
<br />
{{#set:Technical service=DatLab}}<br />
<br />
<br />
[[Category:Technical service]]<br />
<br />
<br />
<br />
<br />
{{Technical service}}<br />
'''Problem:'''<br />
Plotting of O2 flux per mass\ unit sample or flow per cell don't work - either via the manual selecting of the plot under menu 'Graph - Select Plots' or by choosing the appropriate layout under menu 'Layout'.<br />
<br />
'''Cause:'''<br />
<br />
# Sample Concentration\Amonut inside the 'Edit Experiment' window (menu 'Experiment - Edit') equals to 0<br />
# The information of the Sample Unit\Concentration\Amonut inside the 'Edit Experiment' window (menu 'Experiment - Edit') hasn't been saved yet. Although the values inside this window are taken from the last experiment (measured with this DatLab installation, where the 'Save' button inside the 'Edit Experiment' has been pressed) and displayed at the begginig of a next experiments, they're not automatically saved.<br />
<br />
'''Solution:'''<br />
<br />
# Enter the Concentration\Amonut different from 0<br />
# Inside the 'Edit Experiment' window (menu 'Experiment - Edit'), review (or enter) the sample information (Unit\Concentration\Amonut) click on the 'Save' button instead of 'Cancel'. Then (if the Concentration\Amonut not equals to 0) the appropriate plot can be selected.<br />
<br />
[[User:Capek Ondrej|Capek Ondrej]] 15:24, 2 April 2015 (CEST)<br />
<br />
<br />
{{#set:Technical service=DatLab}}<br />
<br />
__SHOWFACTBOX__<br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Control_lights&diff=89623Talk:Control lights2015-05-19T14:14:26Z<p>Harrison DK: </p>
<hr />
<div>{{Technical support}}<br />
'''The meaning of the control LEDs on the oxygraph:'''<br />
<br />
On the left hand side: the lights should be green <br />
<br />
On the right hand side: the 'comm' light should be yellow or green <br />
<br />
Left and right stirrer: should be red, yellow or green.<br />
<br />
<br />
Should these lights be unlit, you should exchange the fuse on the rear of the O2k where the power cable is plugged in. (A spare one is supplied with the O2k in the accessory box). If the 'comm' light is red, this indicates an electronic problem. <br />
<br />
<br />
{{#set:Technical service=Main unit}}<br />
<br />
<br />
<br />
[[Category:Technical service]]<br />
<br />
<br />
<br />
____<br />
<br />
{{Technical service}}<br />
'''Meaning of the control LEDs on the oxygraph:'''<br />
<br />
On the left: lights should be green, <br />
On the right: the 'comm' light should be yellow or green, <br />
Stirrer left and right: should be red, yellow or green.<br />
Are these lights on?<br />
<br />
If not, you should exchange the fuse (a spare should be in the accessory box) on the rear of the O2k where the power cable is plugged in. If the 'comm' light is red then an electronic problem is indicated. <br />
<br />
<br />
{{#set:Technical service=Main unit}}<br />
<br />
__SHOWFACTBOX__<br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Cleaning_the_POS_connector&diff=89617Talk:Cleaning the POS connector2015-05-19T14:11:23Z<p>Harrison DK: </p>
<hr />
<div>{{Technical service}}<br />
<br />
__TOC__<br />
<br />
For detailed information see 'Cleaning of the Electrical Connection' in the POS Service manual [[MiPNet08.04]].<br />
<br />
The two electronic connections of the POS connector (to the [[POS]] and to the [[Main unit]]) make a POS connector that has been disconnected from the [[POS]] or from the [[Main unit]] or from both particularly sensitive to damage by [[ESD]]. '''It is therefore of primary importance to follow the guidelines for ESD protection [[MiPNet14.1]] whenever handling a POS connector.'''<br />
<br />
==Cleaning the electrical connection ==<br />
Unscrew the [[POS]] and check both sides of the electrical connection (the gold pin and thread both on the POS and on the [[POS connector (technical service)|POS connector)]]. Remove any contamination such as grease and moisture with a fine paper towel. Soak a paper towel in ethanol and clean the gold pin and threads. If the gold pin and thread on the POS connector are discolored or stained repeat cleaning with a paper towel soaked in ethanol: Continue cleaning until a fresh paper towel stays clean. Finally, apply a drop of [[contact oil]] to each of the golden pins and threads on POS connector and POS. (You can find a small of bottle of contact oil in your accessories box). If you have also done a [[Sensor service]] or changed the POS membranes allow the oxygraph to run over night before repeating the Sensor test. If all service precautions fail to solve the problem, a new POS connector has to be applied. <br />
<br />
==Cleaning the spring mechanism==<br />
Media outside the chamber or other spills may get into the mechanical part of the POS connector restricting the operation of the spring mechanism hard. The outside of the POS connector should therefore be cleaned with pure water from time to time.<br />
Apply a POS to the POS connector and make sure its seal (to the connector) is tight. Leave the POS connector attached to the oxygraph main unit or make sure that the plug, which connects the POS connector to the main unit, does not get wet (parafilm, or some other form of shielding). This will ensure that only the outside of the POS connector becomes wet. Then rinse the blue plastic of the connector with pure water, working the spring loaded holder several times (similarly to when inserting the connector into the chamber). This will dissolve any media stuck in the mechanical part of the POS connector.<br />
<br />
{{#set: Technical service=POS connector|Technical service=O2 signal}}<br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Cleaning_the_POS_connector&diff=89613Talk:Cleaning the POS connector2015-05-19T13:57:40Z<p>Harrison DK: </p>
<hr />
<div>{{Technical service}}<br />
<br />
__TOC__<br />
<br />
For detailed information see 'Cleaning of the Electrical Connection' in the POS Service manual [[MiPNet08.04]].<br />
<br />
The two electronic connections of the POS connector (to the [[POS]] and to the [[Main unit]]) make a POS connector that has been disconnected from the [[POS]] or from the [[Main unit]] or from both particularly sensitive to damage by [[ESD]]. '''It is therefore of primary importance to follow the guidelines for ESD protection [[MiPNet14.1]] whenever handling a POS connector.'''<br />
<br />
==Cleaning the electrical connection ==<br />
Unscrew the [[POS]] and check both sides of the electrical connection (the gold pin and thread both on the POS and on the [[POS connector (technical service)|POS connector)]]. Remove any contamination such as grease and moister with a fine paper cloth. Soak a paper cloth in ethanol and clean the gold pin and threads. If the gold pin and thread on the POS connector are discolored or stained repeat cleaning with a paper towel soaked in ethanol: Continue cleaning until a fresh paper towel stays clean! <br />
Finally, apply a drop of [[contact oil]] (you can find a small of bottle contact oil in your accessories box) to each of the golden pins and threads on POS connector and POS.<br />
If you have also done a [[Sensor service]] or changed the POS membranes allow the oxygraph to run over night before repeating the Sensor test. If all service precautions fail to solve the problem, a new POS connector has to be applied. <br />
<br />
==Cleaning the spring mechanism==<br />
##From the outside media or other## spills may get into the mechanical part of the POS connector ##making the spring mechanism hard to work##. Therefore, the outside of the POS connector should be cleaned with pure water from time to time.<br />
Apply a POS to the POS connector and make sure its seal (to the connector) is tight. Leave the POS connector attached to the oxygraph main unit or make sure that the plug, which connects the POS connector to the main unit, does not get wet (parafilm, 'hiding' the cable in your sleeves,...).<br />
This will ensure that only the outside of the POS connector is touched by water. Then rinse the blue plastic of the connector with pure water, ##working the spring loaded handle (like when inserting the connector into the chamber) several times##. This will dissolve any media stuck in the mechanical part of the POS connector.<br />
<br />
{{#set: Technical service=POS connector|Technical service=O2 signal}}<br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Cleaning_the_POS_connector&diff=89612Talk:Cleaning the POS connector2015-05-19T13:54:36Z<p>Harrison DK: </p>
<hr />
<div>{{Technical service}}<br />
<br />
__TOC__<br />
<br />
##For detailed information see 'Cleaning of the Electrical Connection' in the POS Service manual [[MiPNet08.04]].##<br />
<br />
The two electronic connections of the POS connector (to the [[POS]] and to the [[Main unit]]) make a POS connector that has been disconnected from the [[POS]] or from the [[Main unit]] or from both particularly sensitive to damage by [[ESD]]. '''It is therefore of primary importance to follow the guidelines for ESD protection [[MiPNet14.1]] whenever handling a POS connector.'''<br />
<br />
==Cleaning the electrical connection ==<br />
Unscrew the [[POS]] and check both sides of the electrical connection (the gold pin and thread both on the POS and on the [[POS connector (technical service)|POS connector)]]. Remove any contamination such as grease and moister with a fine paper cloth. Soak a paper cloth in ethanol and clean the gold pin and threads. If the gold pin and thread on the POS connector are discolored or stained repeat cleaning with a paper towel soaked in ethanol: Continue cleaning until a fresh paper towel stays clean! <br />
Finally, apply a drop of [[contact oil]] (you can find a small of bottle contact oil in your accessories box) to each of the golden pins and threads on POS connector and POS.<br />
If you have also done a [[Sensor service]] or changed the POS membranes allow the oxygraph to run over night before repeating the Sensor test. If all service precautions fail to solve the problem, a new POS connector has to be applied. <br />
<br />
==Cleaning the spring mechanism==<br />
##From the outside media or other## spills may get into the mechanical part of the POS connector ##making the spring mechanism hard to work##. Therefore, the outside of the POS connector should be cleaned with pure water from time to time.<br />
Apply a POS to the POS connector and make sure its seal (to the connector) is tight. Leave the POS connector attached to the oxygraph main unit or make sure that the plug, which connects the POS connector to the main unit, does not get wet (parafilm, 'hiding' the cable in your sleeves,...).<br />
This will ensure that only the outside of the POS connector is touched by water. Then rinse the blue plastic of the connector with pure water, ##working the spring loaded handle (like when inserting the connector into the chamber) several times##. This will dissolve any media stuck in the mechanical part of the POS connector.<br />
<br />
{{#set: Technical service=POS connector|Technical service=O2 signal}}<br />
<br />
[[Category:Technical service]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=O2k-chamber&diff=89611O2k-chamber2015-05-19T13:40:45Z<p>Harrison DK: </p>
<hr />
<div>{{Product<br />
|description='''O2k-Chamber''': 16 mm inner diameter, Duran glass polished, with standard operation volume (''V'') of 2 cm<sup>3</sup> (2 ml).<br />
<br />
Two units of this item are standard components of the [[O2k-Assembly Kit]] ([[O2k-Core]]). The optical properties of Duran allow application in the [[O2k-Fluorometer]] ([http://www.duran-group.com/en/about-duran/duran-properties/optical-properties-of-duran.html Duran optical properties]).<br />
|product id=23100-01<br />
|product type=O2k, O2k-Assembly Kit<br />
|info=[[MiPNet09.01 O2k-ParadigmShift]]<br />
|product image=[[Image:Glass chamber.jpg|right|180px]]<br />
}}<br />
A spare (third) O2k-Chamber may be added to the items ordered with the O2k-System. For dispatch of the [[Oxygraph-2k|O2k]], the O2k-Chambers are shipped in [[O2k-Service Box]].<br />
<br />
<br />
'''<big><big>O2k-technical support</big></big>'''<br />
__TOC__<br />
<br />
== Cleaning the glass chamber ==<br />
<br />
{{Technical support integrated}}<br />
<br />
: Standard operating procedure - cleaning and storage of the O2k-Chamber: >> [[MiPNet19.03 O2k-cleaning and ISS]]<br />
<br />
<br />
== Chamber assembly ==<br />
<br />
{{Technical support integrated}}<br />
<br />
: The assembly of the chamber is described in [[MiPNet19.18A_O2k-Start]].<br />
<br />
'''Possible problems'''<br />
:* Chamber leaking liquids (medium is leaking out of the chamber)<br />
:* Oxygen leak of the chamber (strongly negative intercept in the [[instrumental background test]]<br />
:* Unstable O2 signal<br />
:* Unexpected high or low O2 signal<br />
<br />
'''Trouble shooting procedure'''<br />
: If the problem is obviously related to the chamber proceed to solutions. If the problem manifests in a very low or zero raw signal, the possible contribution of chamber assembly to the problem is easily checked by doing the first step of [[Locating_a_problem#Remove_components]]: if a very low or zero raw signal changes to the expected values (see [[Raw signal]] the problem is the chamber assembly.<br />
<br />
: Otherwise follow [[Locating a problem]]<br />
: If the problem is not located on the [[POS]] or the [[POS connector]] try solving the problem by reassembling the chamber before concluding that the [[main unit]] is defective.<br />
<br />
'''Solutions'''<br />
:* Disassemble the chamber by reversing the instructions in [[MiPNet19.18A O2k-Start]].<br />
:* Check for visible damages on the [[glass chamber]], on the [[POS seal tip]] and all o-rings (on chamber holders or on stoppers). If damage is visible replace the part or consult OROBOROS Instruments. If there is a deposit visible in the glass chamber see [[Cleaning the glass chamber]].<br />
:* Reassemble the chamber following exactly the description in [[MiPNet19.18A O2k-Start]]. In particular, use only small increments (not more than successive quarter turns of the POS holder) when lowering the chamber.<br />
<br />
<br />
== Chamber volume calibration ==<br />
<br />
{{Technical support integrated}}<br />
<br />
: The volume calibration of the chamber is described in [[MiPNet19.18A_O2k-Start]].<br />
<br />
: Old model stopper: if you use blue Titanium stoppers: check the manual delivered with your O2k or contact OROBOROS INSTRUMENTS ([mailto:instruments@oroboros.at instruments@oroboros.at]) for instructions.<br />
<br />
<br />
== Glass chamber stuck in the O2k ==<br />
<br />
{{Technical support integrated}}<br />
<br />
'''Problem'''<br />
: After removing [[OroboPOS-Connector]], [[OroboPOS-Holder]], and [[O2k-Chamber Holder]] from the O2k , the [[O2k-Chamber]] cannot be removed. It remains stuck even after washing with water.<br />
<br />
'''Solution'''<br />
: The reason for this problem is very likely to be that medium has been spilled and is now acting now as an adhesive between glass chamber and copper block. Wash with plenty of warm water and try to remove the chamber while the O2k is switched on and heated up to 37 °C. You can try GENTLY to use the sensor holder (not the sensor connector) to try to push the glass chamber upwards (like you do during [[chamber assembly]]) - while the O2k is warm and with plenty of warm water. With the sensors removed you can also try using a higher temperature, e.g. 40° C (or even slightly higher) for the washing / removing attempts.<br />
<br />
<br />
{{#set: Technical service =Chamber}}<br />
[[Category:Technical service]]<br />
[[Category:All]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=O2k-Open_Support&diff=89610O2k-Open Support2015-05-19T13:26:35Z<p>Harrison DK: </p>
<hr />
<div>{{OROBOROS navigation line page name}}<br />
[[Image:O2k-Info.png|left|80px|link=OROBOROS info|OROBOROS info]]<br />
<br />
<br />
__TOC__<br />
<br />
== O2k-Manual and O2k-Protocols ==<br />
'''O2k-technical support''' is based on and extends the guidelines provided with the [[Oxygraph-2k| O2k]]:<br />
::» [[O2k-Manual]]<br />
::» [[O2k-Protocols]]<br />
::» [[O2k-Publications: Topics]]<br />
<br />
Standardized instrumental [[quality control]] tests are performed to evaluate and diagnose technical problems fast and cost-effectively.<br />
::» Polarographic oxygen sensors: calibration, accuracy and quality control SOP. »[[MiPNet06.03 POS-Calibration-SOP]]«<br />
::» Instrumental background correction, accuracy of oxygen flux and SOP. »[[MiPNet14.06 InstrumentalBackground]]«<br />
::» O2k-standard operating procedures: »[[O2k-SOP]]«<br />
<br />
== Open innovation and O2k-technical support ==<br />
The OROBOROS-Team welcomes and values feedback from O2k-users and interested scientists. Technical and scientific questions, which are not yet addressed in the available guidelines or are difficult to trace on our website, will be relevant to both specialists and beginners in the field.<br />
<br />
<br />
<big>'''The OROBOROS-Team offers O2k-technical support based on [[Open innovation management |open innovation]]:'''</big> <br />
:: Acceptance of O2k-technical support by the OROBOROS-Team implies the mutual agreement to publish the relevant corresponence or a summary of the correspondence addressing technical and scientific questions on O2k-technology and high-resolution respirometry, including names, institutional and Email addresses. Expansion of [[O2k-SOP]]s is particularly relevant for novel approaches in O2k-Fluorometry and O2k-Spectrophotometry, which are combined in the development of the [[NextGen-O2k]]. '''Open innovation''' extends the support given to individual users to the scientific community and provides feedback to improve the scope and documentation of the O2k-technology - in the spirit of [[Gentle Science]].<br />
<br />
<br />
== O2k-technical service ==<br />
<br />
=== Application problems ===<br />
An '''application problem''' can be diagnosed on the basis of O2k-calibration and instrumental background tests performed in pure incubation medium in the absence of an experimental sample. Analysis of DatLab files recorded during instrumental tests is essential. If proper instrumental performance is obtained by resolving such application problems, any hardware problem is excluded.<br />
<br />
=== Software problems ===<br />
Communication between the O2k and DatLab running on a computer.<br />
: [[DatLab Error Message: Line has wrong length]]: Line has wrong length: This message appears when a signal line has not been transmitted properly from the O2k to DatLab. This is a problem only if the error message appears frequently, and may indicate that the specifications of the laptop are not adequate for proper DatLab performance.<br />
<br />
=== Hardware problems ===<br />
Diagnostic tests are available to identify a defective instrumental component. The two-chamber design of the O2k provides the option to exchange parts between the two chambers and thus localize a defective component: »[[Locating a problem]]<br />
<br />
# Hardware problems that can be solved by user-service: The [[OroboPOS]] (polarographic oxygen sensor) requires user-service at intervals of more than 1 year, and SOPs are available to determine if a sensor service is required at an earlier or later date.<br />
# Hardware problems that require replacement of a defective part.<br />
# Some electronic or mechanical defects may be solved only by service of the O2k in the workshop of OROBOROS INSTRUMENTS, e.g. a defective Peltier unit. <br />
: [[DatLab Error Message: Block temperature]]: Average block temperature over past 10 min has not reached set temperature.<br />
<br />
<br />
== Cost estimation for repair ==<br />
<br />
A cost estimation for repair of the O2k-Main unit due to an electronic/mechanical defect can only be given after inspection of the O2k by our workshop in Austria. Once a cost estimation has been provided, the repair is carried out after the customer’s consent. For shipment of the O2k, follow the [[Shipping an oxygraph |detailed instructions]].<br />
<br />
<br />
== O2k-technical support and open innovation pages ==<br />
<br />
{{#ask: mainlabel=Title|[[Technical service::+]]<br />
| ?Technical service<br />
| limit = 100<br />
}}<br />
<br />
<br />
[[Category:OroboPedia]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Oxygen_flux_in_open_O2k-Chamber&diff=89602Talk:Oxygen flux in open O2k-Chamber2015-05-19T13:03:09Z<p>Harrison DK: </p>
<hr />
<div><br />
__SHOWFACTBOX__<br />
<br />
<br />
<br />
= Mario Fasching =<br />
{{Technical service}}<br />
<br />
<br />
In an [[open chamber]] situation the liquid phase in the chamber is in equilibrium with the atmosphere. All oxygen consumed by the [[POS]] is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the non-background corrected oxygen flux (= first time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab) has to be zero. The background corrected flux is meaningless for the open chamber situation. This is because the background corrected flux corrects for consumption of oxygen by the sensor and diffusion into and out of the closed chamber. Since neither of the latter have any effect on the zero flux in the open chamber situation, an incorrect flux will result when background correction is applied. Only menus that display "O2 slope" uncorrected are suitable for assessing the O2 flux at open chamber. Such menus are: <br />
* 01 Calibration Exp Gr3-Temp<br />
* 02 Background Experiment<br />
* 04 Flux per Volume uncorrected<br />
* Z Trouble Shooting<br />
<br />
The observation of a zero flux with an open chamber is an important performance parameter. It indicates that thermal stability has been reached. Therefore no experiment should be started before a zero oxygen flux has been reached with an open chamber. The suggested criterion for "zero flux" is a flux between <br />
-1 pmol/(s ml) and + 1 pmol (s ml).<br />
<br />
<br />
'''Problem:''' <br />
* The uncorrected slope does not reach the interval +/- 1 pmol/(s ml) even after a prolonged time.<br />
<br />
'''Solutions:'''<br />
* The [[open chamber]] situation may not be ideal. Remove the stoppers completely and create a new gas phase as described in [[open chamber]].<br />
* The slope is always calculated from the calibrated O2 signal. Check the [[POS calibration]]<br />
* Observe the [[Raw signal]] and check if it is in the expected range<br />
* If the raw signal is unstable, and thereby creating an apparent O2 flux, follow the instructions for [[Locating a problem]].<br />
<br />
'''See also:'''<br />
<br />
POS calibration [http://www.oroboros.at/index.php?id=o2k-o2calibration MiPNet12.08]<br />
<br />
O2 Flux analysis [http://www.oroboros.at/index.php?id=o2k-datlab-fluxanalysis MiPNet12.09]<br />
<br />
{{#set:Technical service=O2 signal|Technical service=Chamber}}<br />
<br />
[[Category:Technical service]]<br />
<br />
__SHOWFACTBOX__</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Talk:Oxygen_flux_in_open_O2k-Chamber&diff=89601Talk:Oxygen flux in open O2k-Chamber2015-05-19T13:00:02Z<p>Harrison DK: </p>
<hr />
<div>{{Technical service}}<br />
##Link page with "Flux at closed chamber"##<br />
##headline: Oxygen Flux in open chamber##<br />
<br />
In an [[open chamber]] situation the liquid phase in the chamber is in equilibrium with the atmosphere. All oxygen consumed by the [[POS]] is immediately replaced by the atmosphere. Therefore, the oxygen signal has to be constant and <br />
<br />
##the not background corrected oxygen flux (= first time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab) has to be zero.## (the uncorrected O2 slope in DatLab has to be zero ??)<br />
<br />
<br />
##The background corrected flux has no meaning for an open chamber situation. This is because the background corrected flux corrects for consumption of oxygen by the sensor and diffusion into and out of the closed chamber.## (The corrected O2 slope is of no use in terms of an open chamber situation, especially because it is corrected by the sensor ... ????)<br />
<br />
<br />
<br />
Since both effects have no impact on the zero flux in an open chamber situation, as soon as a background correction is applied to the zero flux of an open chamber situation, a wrong flux will be the consequence. Therefore, only layouts that display uncorrected "O2 slope" are suitable for assessing the O2 flux in an open chamber. Such Layouts are: <br />
* 01 Calibration Exp Gr3-Temp<br />
* 02 Background Experiment<br />
* 04 Flux per Volume uncorrected<br />
* Z Trouble Shooting<br />
<br />
To observe a zero flux in an open chamber is an important performance parameter. It indicates that therm stability has been reached. Therefore, no experiment should be started before a zero oxygen flux in an open chamber has been reached. The suggested criterion for "zero flux" is a flux between <br />
-1 pmol/(s ml) and + 1 pmol (s ml).<br />
<br />
<br />
'''Problem:''' <br />
* The uncorrected slope does not reach the interval +/- 1 pmol/(s ml) even after prolonged waiting.<br />
<br />
'''Solutions:'''<br />
* The [[open chamber]] situation may not be ideal. Remove the stoppers completely and create a new gas phase as described in [[open chamber]].<br />
* The slope is always calculated from the calibrated O2 signal. Check the [[POS calibration]]<br />
* Observe the [[Raw signal]] and check whether it is in the expected range<br />
* If the raw signal is unstable and thereby creating an apparent O2 flux follow the instructions for [[Locating a problem]].<br />
<br />
'''See also:'''<br />
<br />
POS calibration [http://www.oroboros.at/index.php?id=o2k-o2calibration MiPNet12.08]<br />
<br />
O2 Flux analysis [http://www.oroboros.at/index.php?id=o2k-datlab-fluxanalysis MiPNet12.09]<br />
<br />
{{#set:Technical service=O2 signal|Technical service=Chamber}}<br />
<br />
[[Category:Technical service]]<br />
<br />
__SHOWFACTBOX__<br />
<br />
<br />
<br />
= Mario Fasching =<br />
{{Technical service}}<br />
<br />
<br />
In an [[open chamber]] situation the liquid phase in the chamber is in equilibrium with the atmosphere. All oxygen consumed by the [[POS]] is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the non-background corrected oxygen flux (= first time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab) has to be zero. The background corrected flux is meaningless for the open chamber situation. This is because the background corrected flux corrects for consumption of oxygen by the sensor and diffusion into and out of the closed chamber. Since neither of the latter have any effect on the zero flux in the open chamber situation, an incorrect flux will result when background correction is applied. Only menus that display "O2 slope" uncorrected are suitable for assessing the O2 flux at open chamber. Such menus are: <br />
* 01 Calibration Exp Gr3-Temp<br />
* 02 Background Experiment<br />
* 04 Flux per Volume uncorrected<br />
* Z Trouble Shooting<br />
<br />
The observation of a zero flux with an open chamber is an important performance parameter. It indicates that thermal stability has been reached. Therefore no experiment should be started before a zero oxygen flux has been reached with an open chamber. The suggested criterion for "zero flux" is a flux between <br />
-1 pmol/(s ml) and + 1 pmol (s ml).<br />
<br />
<br />
'''Problem:''' <br />
* The uncorrected slope does not reach the interval +/- 1 pmol/(s ml) even after a prolonged time.<br />
<br />
'''Solutions:'''<br />
* The [[open chamber]] situation may not be ideal. Remove the stoppers completely and create a new gas phase as described in [[open chamber]].<br />
* The slope is always calculated from the calibrated O2 signal. Check the [[POS calibration]]<br />
* Observe the [[Raw signal]] and check if it is in the expected range<br />
* If the raw signal is unstable, and thereby creating an apparent O2 flux, follow the instructions for [[Locating a problem]].<br />
<br />
'''See also:'''<br />
<br />
POS calibration [http://www.oroboros.at/index.php?id=o2k-o2calibration MiPNet12.08]<br />
<br />
O2 Flux analysis [http://www.oroboros.at/index.php?id=o2k-datlab-fluxanalysis MiPNet12.09]<br />
<br />
{{#set:Technical service=O2 signal|Technical service=Chamber}}<br />
<br />
[[Category:Technical service]]<br />
<br />
__SHOWFACTBOX__</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Coen_2013_J_Gerontol_A_Biol_Sci_Med_Sci&diff=45325Coen 2013 J Gerontol A Biol Sci Med Sci2013-06-15T10:16:08Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Coen PM, Jubrias SA, Distefano G, Amati F, Mackey DC, Glynn NW, Manini TM, Wohlgemuth SE, Leeuwenburgh C, Cummings SR, Newman AB, Ferrucci L, Toledo FG, Shankland E, Conley KE, Goodpaster BH (2013) Skeletal muscle mitochondrial energetics are associated with maximal aerobic capacity and walking speed in older adults. J Gerontol A Biol Sci Med Sci 68: 447-455.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/?term=23051977 PMID: 23051977]<br />
|authors=Coen PM, Jubrias SA, Distefano G, Amati F, Mackey DC, Glynn NW, Manini TM, Wohlgemuth SE, Leeuwenburgh C, Cummings SR, Newman AB, Ferrucci L, Toledo FG, Shankland E, Conley KE, Goodpaster BH<br />
|year=2013<br />
|journal=J Gerontol A Biol Sci Med Sci<br />
|abstract=BACKGROUND:<br />
Lower ambulatory performance with aging may be related to a reduced oxidative capacity within skeletal muscle. This study examined the associations between skeletal muscle mitochondrial capacity and efficiency with walking performance in a group of older adults.<br />
<br />
METHODS:<br />
Thirty-seven older adults (mean age 78 years; 21 men and 16 women) completed an aerobic capacity (VO2 peak) test and measurement of preferred walking speed over 400 m. Maximal coupled (State 3; St3) mitochondrial respiration was determined by high-resolution respirometry in saponin-permeabilized myofibers obtained from percutanous biopsies of vastus lateralis (n = 22). Maximal phosphorylation capacity (ATPmax) of vastus lateralis was determined in vivo by (31)P magnetic resonance spectroscopy (n = 30). Quadriceps contractile volume was determined by magnetic resonance imaging. Mitochondrial efficiency (max ATP production/max O2 consumption) was characterized using ATPmax per St3 respiration (ATPmax/St3).<br />
<br />
RESULTS:<br />
In vitro St3 respiration was significantly correlated with in vivo ATPmax (r (2) = .47, p = .004). Total oxidative capacity of the quadriceps (St3*quadriceps contractile volume) was a determinant of VO2 peak (r (2) = .33, p = .006). ATPmax (r (2) = .158, p = .03) and VO2 peak (r (2) = .475, p < .0001) were correlated with preferred walking speed. Inclusion of both ATPmax/St3 and VO2 peak in a multiple linear regression model improved the prediction of preferred walking speed (r (2) = .647, p < .0001), suggesting that mitochondrial efficiency is an important determinant for preferred walking speed.<br />
<br />
CONCLUSIONS:<br />
Lower mitochondrial capacity and efficiency were both associated with slower walking speed within a group of older participants with a wide range of function. In addition to aerobic capacity, lower mitochondrial capacity and efficiency likely play roles in slowing gait speed with age<br />
|keywords=Muscle, mitochondria, aging, walking speed<br />
|mipnetlab=US FL Gainesville Wohlgemuth SE, US FL Gainesville Leeuwenburgh C, US PA Pittsburgh Goodpaster BH<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=Aging; Senescence<br />
|organism=Human<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|couplingstates=OXPHOS<br />
|topics=ATP; ADP; AMP; PCr<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Fessel_2013_Am_J_Respir_Cell_Mol_Biol&diff=45324Fessel 2013 Am J Respir Cell Mol Biol2013-06-15T10:06:05Z<p>Harrison DK: </p>
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<div>{{Publication<br />
|title=Fessel JP, Flynn CR, Robinson LJ, Penner NL, Gladson S, Kang C, Wasserman DH, Hemnes AR, West JD (2013) Hyperoxia synergizes with mutant BMPR2 to cause metabolic stress, oxidant injury, and pulmonary hypertension. Am J Respir Cell Mol Biol [Epub ahead of print].<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/23742019 PMID: 23742019]<br />
|authors=Fessel JP, Flynn CR, Robinson LJ, Penner NL, Gladson S, Kang C, Wasserman DH, Hemnes AR, West JD<br />
|year=2013<br />
|journal=Am J Respir Cell Mol Biol<br />
|abstract=Pulmonary arterial hypertension (PAH) has been associated with a number of different but interrelated pathogenic mechanisms. Metabolic and oxidative stresses have been shown to play important pathogenic roles in a variety of model systems. However, many of these relationships remain at the level of association. We sought to establish a direct role for metabolic stress and oxidant injury in the pathogenesis of PAH. Mice that universally express a disease-causing Bmpr2 mutation were exposed to room air or to brief daily hyperoxia (95% oxygen for 3 hours) for 6 weeks and compared to wild-type animals with identical exposures. In both murine tissues and cultured endothelial cells, expression of mutant Bmpr2 was sufficient to cause oxidant injury that was particularly pronounced in mitochondrial membranes. With enhancement of mitochondrial generation of reactive oxygen species by hyperoxia, oxidant injury was substantially enhanced in mitochondrial membranes, even in tissues distant from the lung. Hyperoxia, despite its vasodilatory actions in the pulmonary circulation, significantly worsened the PAH phenotype (elevated RVSP, decreased cardiac output, increased pulmonary vascular occlusion) in Bmpr2 mutant animals. These studies demonstrate that oxidant injury and metabolic stress contribute directly to disease development and provide further evidence for PAH as a systemic disease with life-limiting cardiopulmonary manifestations.<br />
|keywords=Pulmonary arterial hypertension, Bmpr2, hyperoxia<br />
|mipnetlab=US TN Nashville Fessel JP, US TN Nashville Wasserman DH<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=RONS; Oxidative Stress, Genetic Defect; Knockdown; Overexpression<br />
|organism=Mouse<br />
|tissues=Heart, Lung; Gill, Endothelial; Epithelial; Mesothelial Cell<br />
|preparations=Intact Cell; Cultured; Primary<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Kim_2013_PLoS_Genet&diff=45323Kim 2013 PLoS Genet2013-06-15T10:05:16Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Kim S-H, Scott SA, Bennett MJ, Carson RP, Fessel JP, Brown HA, Ess KC (2013) Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency . PLoS Genet 9: e1003563.<br />
|info=[http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1003563 doi:10.1371/journal.pgen.1003563 Open Access]<br />
|authors=Kim S-H, Scott SA, Bennett MJ, Carson RP, Fessel JP, Brown HA, Ess KC<br />
|year=2013<br />
|journal=PLoS Genet<br />
|abstract=Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxavu463) that has an inactivating mutation in the etfa gene. dxavu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxavu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxavu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.<br />
|keywords=Multiple Acyl-CoA dehydrogenase deficiency, mTORC1, multi-organ abnormalities<br />
|mipnetlab=US TN Nashville Fessel JP,<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=Mitochondrial Disease; Degenerative Disease and Defect, Genetic Defect; Knockdown; Overexpression<br />
|organism=Zebrafish<br />
|preparations=Intact Organism<br />
|kinetics=Temperature<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Kiebish_2013_J_Lipid_Res&diff=43569Kiebish 2013 J Lipid Res2013-03-06T17:29:43Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Kiebish MA, Yang K, Liu X, Mancuso DJ, Guan S, Zhao Z, Sims HF, Cerqua R, Cade WT, Han X, Gross RW (2013) Dysfunctional cardiac mitochondrial bioenergetic, lipidomic, and signaling in a murine model of Barth syndrome. J Lipid Res [Epub ahead of print].<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/23410936 PMID: 23410936 Open Access]<br />
|authors=Kiebish MA, Yang K, Liu X, Mancuso DJ, Guan S, Zhao Z, Sims HF, Cerqua R, Cade WT, Han X, Gross RW<br />
|year=2013<br />
|journal=J Lipid Res<br />
|abstract=Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase Tafazzin. Recently, an inducible Tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specific dysregulation of HETEs, prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as deceases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA2γ ablation in Tafazzin deficient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by Tafazzin loss of function. Collectively, we identified specific lipidomic, bioenergetic and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.<br />
|keywords=Lipidome; Cardiolipin; Tafazzin; Phospholipase; Cardiolipin synthase; Electron transport chain<br />
|mipnetlab=US MO St Louis Gross RW,<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=Mitochondrial Disease; Degenerative Disease and Defect, Genetic Defect; Knockdown; Overexpression<br />
|organism=Mouse<br />
|tissues=Heart<br />
|preparations=Isolated Mitochondria<br />
|enzymes=Complex III, Complex V; ATP Synthase<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Piccoli_2013_Hum_Mol_Genet&diff=43439Piccoli 2013 Hum Mol Genet2013-02-27T13:40:29Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Piccoli C, Izzo A, Scrima R, Bonfiglio F, Manco R, Negri R, Quarato G, Cela O, Ripoli M, Prisco M, Gentile F, Calì G, Pinton P, Conti A, Nitsch L, Capitanio N (2013) Chronic pro-oxidative state and mitochondrial dysfunctions are more pronounced in fibroblasts from Down syndrome foeti with congenital heart defects. Hum Mol Genet 22: 1218-1232.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/23257287 PMID: 23257287]<br />
|authors=Piccoli C, Izzo A, Scrima R, Bonfiglio F, Manco R, Negri R, Quarato G, Cela O, Ripoli M, Prisco M, Gentile F, Cali G, Pinton P, Conti A, Nitsch L, Capitanio N<br />
|year=2013<br />
|journal=Hum Mol Genet<br />
|abstract=Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.<br />
|keywords=Down syndrome, congenital heart defect<br />
|mipnetlab=IT Foggia Capitanio N<br />
}}<br />
{{Labeling<br />
|injuries=Genetic Defect; Knockdown; Overexpression<br />
|organism=Human<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Duicu_2013_Can_J_Physiol_Pharmacol&diff=42065Duicu 2013 Can J Physiol Pharmacol2013-02-12T11:17:44Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Duicu OM, Mirica SN, Gheorgheosu DE, Privistirescu AI, Fira-Mladinescu O, Muntean DM (2013) Ageing-induced decrease in cardiac mitochondrial function in healthy rats. Can J Physiol Pharmacol 10.1139/cjpp-2012-0422.<br />
|info=[http://www.nrcresearchpress.com/doi/abs/10.1139/cjpp-2012-0422 10.1139/cjpp-2012-0422]<br />
|authors=Duicu OM, Mirica SN, Gheorgheosu DE, Privistirescu AI, Fira-Mladinescu O, Muntean DM<br />
|year=2013<br />
|journal=Can J Physiol Pharmacol<br />
|abstract=Background: It is widely recognized that mitochondrial dysfunction is a key component of the multifactorial process of ageing. The effects of age on individual components of mitochondrial function vary across species and strains. In the present study we investigated and compared oxygen consumption, membrane potential (Δψ), the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium overload and production of reactive oxygen species (ROS) in heart mitochondria isolated from old vs. adult healthy Sprague-Dawley (SD) rats. Methods. Respirometry studies and Δψ measurements were performed with the Oxygraph-2k (Oroboros, Austria) equipped with a tetraphenylphosphonium electrode. ROS production and calcium retention capacity (CRC) were measured spectrofluorimetrically.<br />
<br />
Results: Our results showed an important decline for all bioenergetic parameters for both complex I and complex II supported-respiration, a decreased Δψ in mitochondria energized with complex I substrates and an increased mitochondrial ROS production in old vs. the adult group. Mitochondrial sensitivity to Ca2+-induced mPTP opening was also increased in the old vs. adult animals. Moreover, the protective effect of cyclosporine A on mPTP opening was significantly reduced in the old group.<br />
<br />
Conclusion: Healthy ageing is associated with heart mitochondria dysfunction in Sprague Dawley rats.<br />
|keywords=Cyclosporine A, heart<br />
|mipnetlab=RO Timisoara Muntean D<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k, TPP, Ca, Spectrofluorometry<br />
|injuries=RONS; Oxidative Stress, Aging; Senescence<br />
|organism=Rat<br />
|tissues=Cardiac muscle<br />
|preparations=Isolated Mitochondria<br />
|substratestates=CI, CII<br />
|enzymes=Complex I, Complex II; Succinate Dehydrogenase<br />
|topics=Membrane Potential<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Lally_2013_PLoS_One&diff=42064Lally 2013 PLoS One2013-02-12T11:04:49Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Lally JS, Herbst EA, Matravadia S, Maher AC, Perry CG, Ventura-Clapier R, Holloway GP (2013) Over-expressing mitofusin-2 in healthy mature mammalian skeletal muscle does not alter mitochondrial bioenergetics. PLoS One 8: e55660.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/23383258 PMID: 23383258]<br />
|authors=Lally JS, Herbst EA, Matravadia S, Maher AC, Perry CG, Ventura-Clapier R, Holloway GP<br />
|year=2013<br />
|journal=PLoS One<br />
|abstract=The role of mitofusin-2 (MFN-2) in regulating mitochondrial dynamics has been well-characterized in lower order eukaryotic cell lines through the complete ablation of MFN-2 protein. However, to support the contractile function of mature skeletal muscle, the subcellular architecture and constituent proteins of this tissue differ substantially from simpler cellular organisms. Such differences may also impact the role of MFN-2 in mature mammalian muscle, and it is unclear if minor fluctuations in MFN-2, as observed in response to physiological perturbations, has a functional consequence. Therefore, we have transiently transfected MFN-2 cDNA into rat tibialis anterior muscle to determine the effect of physiolgically relevant increases in MFN-2 protein on mitochondrial bioenergetics. Permeabilized muscle fibres generated from muscle following MFN-2-transfection were used for functional assessments of mitochondrial bioenergetics. In addition, we have further established a novel method for selecting fibre bundles that are positively transfected, and using this approach transient transfection increased MFN-2 protein ∼2.3 fold in selected muscle fibres. However, this did not alter maximal rates of oxygen consumption or the sensitivity for ADP-stimulated respiration. In addition, MFN-2 over-expression did not alter rates of H(2)O(2) emission. Altogether, and contrary to evidence from lower order cell lines, our results indicate that over-expressing MFN-2 in healthy muscle does not influence mitochondrial bioenergetics in mature mammalian skeletal muscle.<br />
|keywords=Mitofusin-2 (MFN-2), skeletal muscle<br />
|mipnetlab=CA_Guelph_Holloway_GP, CA Toronto Perry CG<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k, Spectrofluorometry<br />
|injuries=RONS; Oxidative Stress<br />
|organism=Rat<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|kinetics=ADP; Pi, Oxygen<br />
|topics=ATP; ADP; AMP; PCr<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Murray_2013_Exp_Physiol&diff=40689Murray 2013 Exp Physiol2013-01-27T15:25:16Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Murray AJ (2013) Of mice and men (and muscle mitochondria). Exp Physiol DOI: 10.1113/expphysiol.2012.071092.<br />
|info=[http://onlinelibrary.wiley.com/doi/10.1113/expphysiol.2012.071092/full DOI: 10.1113/expphysiol.2012.071092]<br />
|authors=Murray AJ<br />
|year=2013<br />
|journal=Exp Physiol<br />
|abstract=For the past decade or more, concerns about the short-term health problems and long-term societal impact of the increasingly obese, sedentary and aged populations of Western countries have fuelled a resurgence in metabolic research. In humans, studies of skeletal muscle mitochondria have been at the forefront of much of this work, because respiratory capacity is highly plastic in skeletal muscle, becoming stimulated following exercise training and caloric restriction or suppressed in response to ageing and hypoxia, with a loss of capacity being strongly associated with fatigue and the aetiology of insulin resistance. The accessibility of muscles such as vastus lateralis, which can be biopsied safely from healthy volunteers, athletes and patients, has allowed functional measures of respiratory capacity and coupling to be made. Alongside this, the timely arrival of genetically manipulated mouse models and sophisticated analytical platforms has allowed the elucidation of intricate mechanisms of metabolic control that modify mitochondrial respiration in response to conditions such as altered substrate and oxygen availability, exercise, inflammation, injury and stress. Experimental animals and, in particular, mouse models have therefore become vital tools in mitochondrial research, yet the suitability of mouse skeletal muscle as a model for human muscle research has recently been questioned owing to interspecies differences in molecular profiles and purported variations in mitochondrial function between muscle types. In this issue of Experimental Physiology, a paper by Jacobs et al. (2013) describes the use of [[high-resolution respirometry]] to address these concerns rigorously. The authors conclude that mouse skeletal muscle mitochondria and, in particular, those of mouse quadriceps closely resemble the mitochondria of human quadriceps (vastus lateralis) with respect to their respiratory capacity and control.<br />
|keywords=Comparative Physiology<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|organism=Human, Mouse<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
}}<br />
When considering the respiratory capacity of a given muscle, studies have frequently focused on mitochondrial density, which, although a major determinant in this regard, does not in itself present a full picture of the bioenergetic profile of the tissue. Mitochondrial populations from different tissues or species are known to be functionally distinct, having different respiration rates for a given unit of mitochondrial mass, and this is by no means a new concept. Indeed, it has been known for over 35 years that even within a single cardiac muscle fibre there are two biochemically distinct mitochondrial subpopulations situated in the subsarcolemmal and intermyofibrillar regions (Palmer et al. 1977). To assess functional differences between such populations, isolated mitochondrial preparations can be useful, yet the process of isolation itself disrupts the mitochondrial reticulum, resulting in malformed organelles, and presents a new problem regarding how best to normalize respiration rates, with protein content often correlating poorly with cristae density. The saponin-permeabilized muscle fibre preparation eliminates the need for a time-consuming and disruptive isolation procedure, retaining an intact mitochondrial network within the myofibre whilst providing access for the direct delivery of substrates and inhibitors to the mitochondria (Kuznetsov et al. 2008).<br />
<br />
The study by Jacobs et al. (2013) utilizes this technique to compare respiration rates per unit mass of muscle from human vastus lateralis with those of murine soleus, gastrocnemius and quadriceps. The authors then corrected these rates to the activity of citrate synthase, which is a marker of skeletal muscle mitochondrial density that has previously been validated in humans (Larsen et al. 2012) and is validated in mice in this paper (Jacobs et al. 2013). As expected, the authors found that the oxidative phosphorylation capacity per unit of muscle mass correlates strongly with mitochondrial content across the three mouse muscles, with the highly oxidative soleus muscle exhibiting the highest citrate synthase activity. When normalized to a unit of mitochondria, however, the authors reveal clear differences in mitochondrial respiratory capacity across the different muscles, with those from gastrocnemius, the most glycolytic muscle, exhibiting the highest rates. Yet despite having lower mass-specific respiration rates than human vastus lateralis, the mitochondria of the mixed-fibre-type mouse quadriceps closely resemble those of human muscle. The authors do, however, note that mitochondrial electron coupling control during fat oxidation in human vastus lateralis is more similar to mouse soleus than it is to mouse quadriceps.<br />
<br />
The paper by Jacobs et al. (2013) therefore demonstrates that the skeletal muscle of healthy, young mice, and mouse quadriceps in particular, is a suitable model tissue for studies of human skeletal muscle mitochondrial function, and these findings will no doubt be met with some relief by the many groups worldwide that utilize mouse models in muscle research. The growing resource of genetically manipulated mouse models with metabolic phenotypes appears to remain a valuable tool for bioenergetic research.<br />
<br />
Perhaps a final word of caution is warranted, however, in that similarities in baseline muscle mitochondrial function between healthy, young, unstressed mice and humans do not suggest that each will necessarily respond in a qualitatively or quantitatively similar fashion to a given pathology or stress. Future studies might therefore follow the methods of Jacobs et al. (2013) to investigate whether the mitochondria of mouse and human quadriceps continue to function in a similar manner when stressed. To this end, comparative studies of hypoxic mouse and human muscle, for instance, would be revealing, because chronic exposure to hypoxia is known to alter muscle mitochondrial density, alongside intramitochondrial changes in respiratory capacity and coupling via altered expression and function of electron transport chain complexes and substrate oxidation enzymes (Murray, 2009).<br />
<br />
Caution should, of course, always be exercised when interpreting animal studies, with care being taken to temper any premature translation into implications for human physiology. The work of Jacobs et al. (2013), however, is important and timely in reasserting the value of the mouse as an experimental model in human metabolic research.<br />
<br />
<br />
'''References'''<br />
<br />
[[Jacobs RA 2012 Exp Physiol|Jacobs RA, Díaz V, Meinild A-K, Gassmann M & Lundby C (2013). The C57Bl/6 mouse serves as a suitable model of human skeletal muscle mitochondrial function. Exp Physiol 98, 000–000.]]<br />
<br />
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R & Kunz WS (2008). Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3, 965–976. <br />
<br />
Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F & Hey-Mogensen M (2012). Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol 590, 3349–3360. <br />
<br />
Murray AJ (2009). Metabolic adaptation of skeletal muscle to high altitude hypoxia: how new technologies could resolve the controversies. Genome Med 1, 117. <br />
<br />
Palmer JW, Tandler B & Hoppel CL (1977). Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252, 8731–8739.</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Singh_2013_J_Cereb_Blood_Flow_Metab&diff=40688Singh 2013 J Cereb Blood Flow Metab2013-01-27T15:18:35Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Singh IN, Gilmer LK, Miller DM, Cebak JE, Wang JA, Hall ED (2013) Phenelzine mitochondrial functional preservation and neuroprotection after traumatic brain injury related to scavenging of the lipid peroxidation-derived aldehyde 4-hydroxy-2-nonenal. J Cereb Blood Flow Metab [Epub ahead of print].<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed?term=Phenelzine%20mitochondrial%20functional%20preservation%20and%20neuroprotection%20after%20traumatic%20brain%20injury%20related%20to%20scavenging%20of%20the%20lipid%20peroxidation-derived%20aldehyde%204-hydroxy-2-nonenal PMID: 23321786]<br />
|authors=Singh IN, Gilmer LK, Miller DM, Cebak JE, Wang JA, Hall ED<br />
|year=2013<br />
|journal=J Cereb Blood Flow Metab<br />
|abstract=Phenelzine (PZ) is a scavenger of the lipid peroxidation (LP)-derived reactive aldehyde 4-hydroxynonenal (4-HNE) due to its hydrazine functional group, which can covalently react with 4-HNE. In this study, we first examined the ability of PZ to prevent the respiratory depressant effects of 4-HNE on normal isolated brain cortical mitochondria. Second, in rats subjected to controlled cortical impact traumatic brain injury (CCI-TBI), we evaluated PZ (10 mg/kg subcutaneously at 15 minutes after CCI-TBI) to attenuate 3-hour post-TBI mitochondrial respiratory dysfunction, and in separate animals, to improve cortical tissue sparing at 14 days. While 4-HNE exposure inhibited mitochondrial complex I and II respiration in a concentration-dependent manner, pretreatment with equimolar concentrations of PZ antagonized these effects. Western blot analysis demonstrated a PZ decrease in 4-HNE in mitochondrial proteins. Mitochondria isolated from peri-contusional brain tissue of CCI-TBI rats treated with vehicle at 15 minutes after injury showed a 37% decrease in the respiratory control ratio ([[RCR]]) relative to noninjured mitochondria. In PZ-treated rats, RCR suppression was prevented (P<0.05 versus vehicle). In another cohort, PZ administration increased spared cortical tissue from 86% to 97% (P<0.03). These results suggest that PZ's neuroprotective effect is due to mitochondrial protection by scavenging of LP-derived 4-HNE.<br />
|keywords=Phenelzine, Traumatic brain injury, Mitochondrial respiratory dysfunction,<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|organism=Rat<br />
|tissues=Neurons; Brain<br />
|preparations=Isolated Mitochondria<br />
|substratestates=CI, CII<br />
|enzymes=Complex I, Complex II; Succinate Dehydrogenase<br />
|topics=Fatty Acid<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Murray_2013_Exp_Physiol&diff=40687Murray 2013 Exp Physiol2013-01-27T15:13:47Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Murray AJ (2013) Of mice and men (and muscle mitochondria). Exp Physiol DOI: 10.1113/expphysiol.2012.071092.<br />
|info=[http://onlinelibrary.wiley.com/doi/10.1113/expphysiol.2012.071092/full DOI: 10.1113/expphysiol.2012.071092]<br />
|authors=Murray AJ<br />
|year=2013<br />
|journal=Exp Physiol<br />
|abstract=For the past decade or more, concerns about the short-term health problems and long-term societal impact of the increasingly obese, sedentary and aged populations of Western countries have fuelled a resurgence in metabolic research. In humans, studies of skeletal muscle mitochondria have been at the forefront of much of this work, because respiratory capacity is highly plastic in skeletal muscle, becoming stimulated following exercise training and caloric restriction or suppressed in response to ageing and hypoxia, with a loss of capacity being strongly associated with fatigue and the aetiology of insulin resistance. The accessibility of muscles such as vastus lateralis, which can be biopsied safely from healthy volunteers, athletes and patients, has allowed functional measures of respiratory capacity and coupling to be made. Alongside this, the timely arrival of genetically manipulated mouse models and sophisticated analytical platforms has allowed the elucidation of intricate mechanisms of metabolic control that modify mitochondrial respiration in response to conditions such as altered substrate and oxygen availability, exercise, inflammation, injury and stress. Experimental animals and, in particular, mouse models have therefore become vital tools in mitochondrial research, yet the suitability of mouse skeletal muscle as a model for human muscle research has recently been questioned owing to interspecies differences in molecular profiles and purported variations in mitochondrial function between muscle types. In this issue of Experimental Physiology, a paper by Jacobs et al. (2013) describes the use of [[high-resolution respirometry]] to address these concerns rigorously. The authors conclude that mouse skeletal muscle mitochondria and, in particular, those of mouse quadriceps closely resemble the mitochondria of human quadriceps (vastus lateralis) with respect to their respiratory capacity and control.<br />
|keywords=Comparative Physiology, Skeletal muscle, Mouse, Human<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|organism=Human, Mouse<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
}}<br />
When considering the respiratory capacity of a given muscle, studies have frequently focused on mitochondrial density, which, although a major determinant in this regard, does not in itself present a full picture of the bioenergetic profile of the tissue. Mitochondrial populations from different tissues or species are known to be functionally distinct, having different respiration rates for a given unit of mitochondrial mass, and this is by no means a new concept. Indeed, it has been known for over 35 years that even within a single cardiac muscle fibre there are two biochemically distinct mitochondrial subpopulations situated in the subsarcolemmal and intermyofibrillar regions (Palmer et al. 1977). To assess functional differences between such populations, isolated mitochondrial preparations can be useful, yet the process of isolation itself disrupts the mitochondrial reticulum, resulting in malformed organelles, and presents a new problem regarding how best to normalize respiration rates, with protein content often correlating poorly with cristae density. The saponin-permeabilized muscle fibre preparation eliminates the need for a time-consuming and disruptive isolation procedure, retaining an intact mitochondrial network within the myofibre whilst providing access for the direct delivery of substrates and inhibitors to the mitochondria (Kuznetsov et al. 2008).<br />
<br />
The study by Jacobs et al. (2013) utilizes this technique to compare respiration rates per unit mass of muscle from human vastus lateralis with those of murine soleus, gastrocnemius and quadriceps. The authors then corrected these rates to the activity of citrate synthase, which is a marker of skeletal muscle mitochondrial density that has previously been validated in humans (Larsen et al. 2012) and is validated in mice in this paper (Jacobs et al. 2013). As expected, the authors found that the oxidative phosphorylation capacity per unit of muscle mass correlates strongly with mitochondrial content across the three mouse muscles, with the highly oxidative soleus muscle exhibiting the highest citrate synthase activity. When normalized to a unit of mitochondria, however, the authors reveal clear differences in mitochondrial respiratory capacity across the different muscles, with those from gastrocnemius, the most glycolytic muscle, exhibiting the highest rates. Yet despite having lower mass-specific respiration rates than human vastus lateralis, the mitochondria of the mixed-fibre-type mouse quadriceps closely resemble those of human muscle. The authors do, however, note that mitochondrial electron coupling control during fat oxidation in human vastus lateralis is more similar to mouse soleus than it is to mouse quadriceps.<br />
<br />
The paper by Jacobs et al. (2013) therefore demonstrates that the skeletal muscle of healthy, young mice, and mouse quadriceps in particular, is a suitable model tissue for studies of human skeletal muscle mitochondrial function, and these findings will no doubt be met with some relief by the many groups worldwide that utilize mouse models in muscle research. The growing resource of genetically manipulated mouse models with metabolic phenotypes appears to remain a valuable tool for bioenergetic research.<br />
<br />
Perhaps a final word of caution is warranted, however, in that similarities in baseline muscle mitochondrial function between healthy, young, unstressed mice and humans do not suggest that each will necessarily respond in a qualitatively or quantitatively similar fashion to a given pathology or stress. Future studies might therefore follow the methods of Jacobs et al. (2013) to investigate whether the mitochondria of mouse and human quadriceps continue to function in a similar manner when stressed. To this end, comparative studies of hypoxic mouse and human muscle, for instance, would be revealing, because chronic exposure to hypoxia is known to alter muscle mitochondrial density, alongside intramitochondrial changes in respiratory capacity and coupling via altered expression and function of electron transport chain complexes and substrate oxidation enzymes (Murray, 2009).<br />
<br />
Caution should, of course, always be exercised when interpreting animal studies, with care being taken to temper any premature translation into implications for human physiology. The work of Jacobs et al. (2013), however, is important and timely in reasserting the value of the mouse as an experimental model in human metabolic research.<br />
<br />
<br />
'''References'''<br />
<br />
[[Jacobs RA 2012 Exp Physiol|Jacobs RA, Díaz V, Meinild A-K, Gassmann M & Lundby C (2013). The C57Bl/6 mouse serves as a suitable model of human skeletal muscle mitochondrial function. Exp Physiol 98, 000–000.]]<br />
<br />
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R & Kunz WS (2008). Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3, 965–976. <br />
<br />
Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F & Hey-Mogensen M (2012). Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol 590, 3349–3360. <br />
<br />
Murray AJ (2009). Metabolic adaptation of skeletal muscle to high altitude hypoxia: how new technologies could resolve the controversies. Genome Med 1, 117. <br />
<br />
Palmer JW, Tandler B & Hoppel CL (1977). Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252, 8731–8739.</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Murray_2013_Exp_Physiol&diff=40686Murray 2013 Exp Physiol2013-01-27T15:12:47Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Murray AJ (2013) Of mice and men (and muscle mitochondria). Exp Physiol DOI: 10.1113/expphysiol.2012.071092.<br />
|info=[http://onlinelibrary.wiley.com/doi/10.1113/expphysiol.2012.071092/full DOI: 10.1113/expphysiol.2012.071092]<br />
|authors=Murray AJ<br />
|year=2013<br />
|journal=Exp Physiol<br />
|abstract=For the past decade or more, concerns about the short-term health problems and long-term societal impact of the increasingly obese, sedentary and aged populations of Western countries have fuelled a resurgence in metabolic research. In humans, studies of skeletal muscle mitochondria have been at the forefront of much of this work, because respiratory capacity is highly plastic in skeletal muscle, becoming stimulated following exercise training and caloric restriction or suppressed in response to ageing and hypoxia, with a loss of capacity being strongly associated with fatigue and the aetiology of insulin resistance. The accessibility of muscles such as vastus lateralis, which can be biopsied safely from healthy volunteers, athletes and patients, has allowed functional measures of respiratory capacity and coupling to be made. Alongside this, the timely arrival of genetically manipulated mouse models and sophisticated analytical platforms has allowed the elucidation of intricate mechanisms of metabolic control that modify mitochondrial respiration in response to conditions such as altered substrate and oxygen availability, exercise, inflammation, injury and stress. Experimental animals and, in particular, mouse models have therefore become vital tools in mitochondrial research, yet the suitability of mouse skeletal muscle as a model for human muscle research has recently been questioned owing to interspecies differences in molecular profiles and purported variations in mitochondrial function between muscle types. In this issue of Experimental Physiology, a paper by Jacobs et al. (2013) describes the use of [[high-resolution respirometry]] to address these concerns rigorously. The authors conclude that mouse skeletal muscle mitochondria and, in particular, those of mouse quadriceps closely resemble the mitochondria of human quadriceps (vastus lateralis) with respect to their respiratory capacity and control.<br />
|keywords=Comparative Physiology, skeletal muscle, mouse, human<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|organism=Human, Mouse<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
}}<br />
When considering the respiratory capacity of a given muscle, studies have frequently focused on mitochondrial density, which, although a major determinant in this regard, does not in itself present a full picture of the bioenergetic profile of the tissue. Mitochondrial populations from different tissues or species are known to be functionally distinct, having different respiration rates for a given unit of mitochondrial mass, and this is by no means a new concept. Indeed, it has been known for over 35 years that even within a single cardiac muscle fibre there are two biochemically distinct mitochondrial subpopulations situated in the subsarcolemmal and intermyofibrillar regions (Palmer et al. 1977). To assess functional differences between such populations, isolated mitochondrial preparations can be useful, yet the process of isolation itself disrupts the mitochondrial reticulum, resulting in malformed organelles, and presents a new problem regarding how best to normalize respiration rates, with protein content often correlating poorly with cristae density. The saponin-permeabilized muscle fibre preparation eliminates the need for a time-consuming and disruptive isolation procedure, retaining an intact mitochondrial network within the myofibre whilst providing access for the direct delivery of substrates and inhibitors to the mitochondria (Kuznetsov et al. 2008).<br />
<br />
The study by Jacobs et al. (2013) utilizes this technique to compare respiration rates per unit mass of muscle from human vastus lateralis with those of murine soleus, gastrocnemius and quadriceps. The authors then corrected these rates to the activity of citrate synthase, which is a marker of skeletal muscle mitochondrial density that has previously been validated in humans (Larsen et al. 2012) and is validated in mice in this paper (Jacobs et al. 2013). As expected, the authors found that the oxidative phosphorylation capacity per unit of muscle mass correlates strongly with mitochondrial content across the three mouse muscles, with the highly oxidative soleus muscle exhibiting the highest citrate synthase activity. When normalized to a unit of mitochondria, however, the authors reveal clear differences in mitochondrial respiratory capacity across the different muscles, with those from gastrocnemius, the most glycolytic muscle, exhibiting the highest rates. Yet despite having lower mass-specific respiration rates than human vastus lateralis, the mitochondria of the mixed-fibre-type mouse quadriceps closely resemble those of human muscle. The authors do, however, note that mitochondrial electron coupling control during fat oxidation in human vastus lateralis is more similar to mouse soleus than it is to mouse quadriceps.<br />
<br />
The paper by Jacobs et al. (2013) therefore demonstrates that the skeletal muscle of healthy, young mice, and mouse quadriceps in particular, is a suitable model tissue for studies of human skeletal muscle mitochondrial function, and these findings will no doubt be met with some relief by the many groups worldwide that utilize mouse models in muscle research. The growing resource of genetically manipulated mouse models with metabolic phenotypes appears to remain a valuable tool for bioenergetic research.<br />
<br />
Perhaps a final word of caution is warranted, however, in that similarities in baseline muscle mitochondrial function between healthy, young, unstressed mice and humans do not suggest that each will necessarily respond in a qualitatively or quantitatively similar fashion to a given pathology or stress. Future studies might therefore follow the methods of Jacobs et al. (2013) to investigate whether the mitochondria of mouse and human quadriceps continue to function in a similar manner when stressed. To this end, comparative studies of hypoxic mouse and human muscle, for instance, would be revealing, because chronic exposure to hypoxia is known to alter muscle mitochondrial density, alongside intramitochondrial changes in respiratory capacity and coupling via altered expression and function of electron transport chain complexes and substrate oxidation enzymes (Murray, 2009).<br />
<br />
Caution should, of course, always be exercised when interpreting animal studies, with care being taken to temper any premature translation into implications for human physiology. The work of Jacobs et al. (2013), however, is important and timely in reasserting the value of the mouse as an experimental model in human metabolic research.<br />
<br />
<br />
'''References'''<br />
<br />
[[Jacobs RA 2012 Exp Physiol|Jacobs RA, Díaz V, Meinild A-K, Gassmann M & Lundby C (2013). The C57Bl/6 mouse serves as a suitable model of human skeletal muscle mitochondrial function. Exp Physiol 98, 000–000.]]<br />
<br />
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R & Kunz WS (2008). Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3, 965–976. <br />
<br />
Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F & Hey-Mogensen M (2012). Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol 590, 3349–3360. <br />
<br />
Murray AJ (2009). Metabolic adaptation of skeletal muscle to high altitude hypoxia: how new technologies could resolve the controversies. Genome Med 1, 117. <br />
<br />
Palmer JW, Tandler B & Hoppel CL (1977). Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252, 8731–8739.</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Lou_2013_Cardiovasc_Res&diff=40685Lou 2013 Cardiovasc Res2013-01-27T15:08:03Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Lou PH, Zhang L, Lucchinetti E, Heck M, Affolter A, Gandhi M, Kienesberger PC, Hersberger M, Clanachan AS, Zaugg M (2013) Infarct-remodelled hearts with limited oxidative capacity boost fatty acid oxidation after conditioning against ischaemia/reperfusion injury. Cardiovasc Res 97: 251-261.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed?term=Infarct-remodelled%20hearts%20with%20limited%20oxidative%20capacity%20boost%20fatty%20acid%20oxidation%20after%20conditioning%20against%20ischaemia%2Freperfusion%20injury PMID: 23097573]<br />
|authors=Lou PH, Zhang L, Lucchinetti E, Heck M, Affolter A, Gandhi M, Kienesberger PC, Hersberger M, Clanachan AS, Zaugg M<br />
|year=2013<br />
|journal=Cardiovasc Res<br />
|abstract=''AIMS'': Infarct-remodelled hearts are less amenable to protection against ischaemia/reperfusion. Understanding preservation of energy metabolism in diseased vs. healthy hearts may help to develop anti-ischaemic strategies effective also in jeopardized myocardium.<br />
<br />
''METHODS AND RESULTS'': Isolated infarct-remodelled/sham Sprague-Dawley rat hearts were perfused in the working mode and subjected to 15 min of ischaemia and 30 min of reperfusion. Protection of post-ischaemic ventricular work was achieved by pharmacological conditioning with sevoflurane. Oxidative metabolism was measured by substrate flux in fatty acid and glucose oxidation using [(3)H]palmitate and [(14)C]glucose. Mitochondrial oxygen consumption was measured in saponin-permeabilized left ventricular muscle fibres. Activity assays of citric acid synthase, hydroxyacyl-CoA dehydrogenase, and pyruvate dehydrogenase and mass spectrometry for acylcarnitine profiling were also performed. Six weeks after coronary artery ligation, the hearts exhibited macroscopic and molecular signs of hypertrophy consistent with remodelling and limited respiratory chain and citric acid cycle capacity. Unprotected remodelled hearts showed a marked decline in palmitate oxidation and acetyl-CoA energy production after ischaemia/reperfusion, which normalized in sevoflurane-protected remodelled hearts. Protected remodelled hearts also showed higher β-oxidation flux as determined by increased oxygen consumption with palmitoylcarnitine/malate in isolated fibres and a lower ratio of C16:1+C16OH/C14 carnitine species, indicative of a higher long-chain hydroxyacyl-CoA dehydrogenase activity. Remodelled hearts exhibited higher PPARα-PGC-1α but defective HIF-1α signalling, and conditioning enabled them to mobilize fatty acids from endogenous triglyceride stores, which closely correlated with improved recovery.<br />
<br />
''CONCLUSIONS'': Protected infarct-remodelled hearts secure post-ischaemic energy production by activation of β-oxidation and mobilization of fatty acids from endogenous triglyceride stores.<br />
|keywords=Ischaemia/reperfusion injury, Anti-ischaemic strategies, Sevoflurane, Palmitate oxidation, PPARα-PGC-1α, HIF-1α<br />
|mipnetlab=CA Edmonton Zaugg M<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=Ischemia-Reperfusion; Preservation<br />
|organism=Rat<br />
|tissues=Cardiac muscle<br />
|preparations=Permeabilized tissue<br />
|couplingstates=OXPHOS<br />
|enzymes=TCA Cycle and Matrix Dehydrogenases<br />
|topics=Substrate; Glucose; TCA Cycle, Fatty Acid<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Kristensen_2013_PLoS_One&diff=40684Kristensen 2013 PLoS One2013-01-27T14:59:28Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Kristensen JM, Larsen S, Helge JW, Dela F, Wojtaszewski JFP (2013) Two weeks of metformin treatment enhances mitochondrial respiration in skeletal muscle of AMPK kinase dead but not wild type mice. PLoS One PLoS ONE 8: e53533.<br />
|info=[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0053533 doi:10.1371/journal.pone.0053533 Open Access]<br />
|authors=Kristensen JM, Larsen S, Helge JW, Dela F, Wojtaszewski JFP<br />
|year=2013<br />
|journal=PLoS One<br />
|abstract=Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α2 (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.<br />
<br />
We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.<br />
|keywords=Metformin, 5′AMP activated protein kinase (AMPK), Type 2 diabetes,<br />
|mipnetlab=DK Copenhagen Dela F,<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|organism=Mouse<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|couplingstates=LEAK, OXPHOS<br />
|substratestates=CI, CII, CI+II<br />
|enzymes=Complex I, Complex II; Succinate Dehydrogenase, Complex IV; Cytochrome c Oxidase<br />
|kinetics=Reduced Substrate; Cytochrome c<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Kelly_2013_Stem_Cells&diff=40683Kelly 2013 Stem Cells2013-01-27T14:55:20Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Kelly RD, Rodda AE, Dickinson A, Mahmud A, Nefzger CM, Lee W, Forsythe J, Polo JM, Trounce IA, McKenzie M, Nisbet DR, St John JC (2013) Mitochondrial DNA haplotypes define gene expression patterns in pluripotent and differentiating embryonic stem cells. Stem Cells [Epub ahead of print].<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed?term=Mitochondrial%20DNA%20haplotypes%20define%20gene%20expression%20patterns%20in%20pluripotent%20and%20differentiating%20embryonic%20stem%20cells PMID: 23307500]<br />
|authors=Kelly RD, Rodda AE, Dickinson A, Mahmud A, Nefzger CM, Lee W, Forsythe J, Polo JM, Trounce IA, McKenzie M, Nisbet DR, St John JC<br />
|year=2013<br />
|journal=Stem Cells<br />
|abstract=Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations and ageing. Accumulating evidence suggests that mitochondrial DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype-specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell.<br />
|keywords=Mitochondrial DNA, Haplotype, Phenotype,<br />
|mipnetlab=AU Melbourne Trounce IA,<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=Mitochondrial Disease; Degenerative Disease and Defect, Aging; Senescence, Genetic Defect; Knockdown; Overexpression<br />
|organism=Mouse<br />
|tissues=Cardiac muscle<br />
|couplingstates=OXPHOS<br />
|topics=ATP; ADP; AMP; PCr<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Harrison_2012_Abstract_Bioblast&diff=37178Harrison 2012 Abstract Bioblast2012-11-25T09:11:32Z<p>Harrison DK: </p>
<hr />
<div>{{Abstract<br />
|title=Harrison DK, Fasching M (2012) O2k-Spectrophotometry – A ''MitoCom'' Project. Mitochondr Physiol Network 17.12.<br />
|info=[[MiPNet17.12 Bioblast 2012|MiPNet17.12 Bioblast 2012 - Open Access]]<br />
|authors=Harrison DK, Fasching M,<br />
|year=2012<br />
|event=[[Bioblast 2012]]<br />
|abstract=[[File:DKH ISPS.jpg|right|200px|David Harrison]]<br />
The principal thrust of the technical development of the O2k high-resolution respirometer within the ''MitoCom'' project is towards the O2k Fluorometer. However, simultaneously, a parallel project is working towards the integration of spectrophotometry into the O2k in order to measure the redox state of cytochromes – principally cytochrome c and aa3, but also cytochrome b. <br />
<br />
The first approach to measuring cytochrome absorption spectra in the O2k respirometer used a specially designed stopper that incorporated two lightguide fibres: one used to conduct light from an external lamp into the cuvette, the other to collect the emergent light scattered by the medium [1].<br />
<br />
However, the development of multiple parameter sensors that can be inserted through the conventional stoppers led to the concept of using the chamber window as the optical port-hole for both fluorometry and spectrophotometry [2] instead of the special optical stopper (which could not be used in conjunction with other probes). The initial spectrophotometric approach was to use a pair of lightguides: the first to transmit light through the chamber window and the second to receive the light scattered back from the medium. A series of experiments was carried out in order to determine the optimal configuration and spacing for the lightguides. In order to carry out comparisons, it was necessary to develop analytical methods in order to compare the signal-to-noise (S/N) ratios of the difference spectra (reduced minus oxidised cytochrome absorption spectra) obtained using the different configurations and the results will be presented.<br />
<br />
The latest approach involves incorporation of a white light emitting diode (LED) into the O2k itself, in addition to the standard illuminating LED. Initial results using this configuration demonstrate an order-of-magnitude increase in S/N ratio compared with the previous configurations (Fig. 1). Work is continuing in order to determine the optimal spectral characteristics for the LED.<br />
<br />
(1) [[Sommer 2010 Eur Respir J|Sommer N, Pak O, Schoerner S, Derfuss T, Krug A, Gnaiger E, Ghofrani HA, Schermuly RT, Huckstorf C, Seeger W, Grimminger F, Weissmann N (2010) Mitochondrial cytochrome redox states and respiration in acute pulmonary oxygen sensing. Eur Respir J 36: 1056-1066.]]<br />
<br />
(2) [[Hickey 2012 J Comp Physiol B|Hickey AJ, Renshaw GM, Speers-Roesch B, Richards JG, Wang Y, Farrell AP, Brauner CJ (2012) A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (''Hemiscyllum ocellatum''). J Comp Physiol B 182: 91-100.]]<br />
|keywords=[[Oxygraph-2k]], [[High-resolution respirometry]], Spectrophotometry, ''MitoCom'',<br />
|mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E,<br />
|journal=Mitochondr Physiol Network<br />
|articletype=Abstract<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k, Spectrophotometry<br />
|injuries=Cancer; Apoptosis; Cytochrome c<br />
|organism=Yeast; Fungi<br />
|enzymes=Complex IV; Cytochrome c Oxidase<br />
|kinetics=Reduced Substrate; Cytochrome c<br />
|journal=Mitochondr Physiol Network<br />
|articletype=Abstract<br />
}}<br />
__NOTOC__<br />
<br />
<br />
== Affiliations and author contributions ==<br />
<br />
Oroboros Instruments, Innsbruck, AT<br />
<br />
== Figure 1 ==<br />
[[File:DKH Fig 1.jpg|600px|S/N ratios]]<br />
<br />
Figure 1: Left to Right – S/N ratios for 3mm lightguide separation through window, internal LED with black stirrer, internal LED with white stirrer, stopper cuvette.<br />
<br />
== Help ==<br />
* [[Abstracts help]]<br />
* [[MitoPedia Glossary: Terms and abbreviations]]</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Perry_2012_J_Physiol&diff=36516Perry 2012 J Physiol2012-11-15T14:25:37Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Perry CG, Kane DA, Herbst EA, Mukai K, Lark DS, Wright DC, Heigenhauser GJ, Neufer PD, Spriet LL, Holloway GP (2012) Mitochondrial creatine kinase activity and phosphate shuttling are acutely regulated by exercise in human skeletal muscle. J Physiol 590: 5475-5486.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/22907058 PMID: 22907058]<br />
|authors=Perry CG, Kane DA, Herbst EA, Mukai K, Lark DS, Wright DC, Heigenhauser GJ, Neufer PD, Spriet LL, Holloway GP<br />
|year=2012<br />
|journal=J Physiol<br />
|abstract=Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving mitochondrial creatine kinase (miCK). However, ADP/ATP diffusion through adenine nucleotide translocase ([[ANT]]) and voltage-dependent anion carriers (VDACs) is also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFBs) taken from biopsies before and after 2 h of cycling exercise (60% ) in nine lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFBs were manipulated ''in situ''. In the absence of contractile signals (relaxed PmFBs) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced ''in situ'' (up to 126% higher apparent K(m) to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDACs). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given that the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDACs) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFBs, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent K(m) to ADP), suggesting prior exercise increases miCK sensitivity ''in situ''. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial-cytosolic energy exchange via PCr/Cr cycling, possibly through VDACs. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient coupling of oxidative phosphorylation to perturbations in cellular energy charge during subsequent bouts of contraction.<br />
|keywords=Post-exercise respiratory sensitivity, creatine kinase, phosphate shuttling, skeletal muscle<br />
}}<br />
{{Labeling<br />
|organism=Human<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|couplingstates=OXPHOS<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Smith_2012_Biochem_J&diff=36515Smith 2012 Biochem J2012-11-15T14:23:09Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Smith BK, Perry CG, Koves TR, Wright DC, Smith JC, Neufer PD, Muoio DM, Holloway GP (2012) Identification of a novel malonyl-CoA IC50 for CPT-I: implications for predicting ''in vivo'' fatty acid oxidation rates. Biochem J 448: 13-20.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed/22928974 PMID: 22928974]<br />
|authors=Smith BK, Perry CG, Koves TR, Wright DC, Smith JC, Neufer PD, Muoio DM, Holloway GP<br />
|year=2012<br />
|journal=Biochem J<br />
|abstract=Published values regarding the sensitivity (IC50) of CPT-I (carnitine palmitoyltransferase I) to M-CoA (malonyl-CoA) inhibition in isolated mitochondria are inconsistent with predicted ''in vivo'' rates of fatty acid oxidation. Therefore we have re-examined M-CoA inhibition kinetics under various P-CoA (palmitoyl-CoA) concentrations in both isolated mitochondria and PMFs (permeabilized muscle fibres). PMFs have an 18-fold higher IC50 (0.61 compared with 0.034 μM) in the presence of 25 μM P-CoA and a 13-fold higher IC50 (6.3 compared with 0.49 μM) in the presence of 150 μM P-CoA compared with isolated mitochondria. M-CoA inhibition kinetics determined in PMFs predicts that CPT-I activity is inhibited by 33% in resting muscle compared with >95% in isolated mitochondria. Additionally, the ability of M-CoA to inhibit CPT-I appears to be dependent on P-CoA concentration, as the relative inhibitory capacity of M-CoA is decreased with increasing P-CoA concentrations. Altogether, the use of PMFs appears to provide an M-CoA IC50 that better reflects the predicted in vivo rates of fatty acid oxidation. These findings also demonstrate that the ratio of [P-CoA]/[M-CoA] is critical for regulating CPT-I activity and may partially rectify the ''in vivo'' disconnect between M-CoA content and CPT-I flux within the context of exercise and Type 2 diabetes.<br />
|keywords=Type 2 diabetes, skeletal muscle, fatty acid oxidation<br />
|mipnetlab=CA Guelph Holloway GP<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|organism=Rat<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue, Isolated Mitochondria<br />
|couplingstates=LEAK, OXPHOS<br />
|topics=Fatty Acid<br />
}}</div>Harrison DKhttps://wiki.oroboros.at/index.php?title=Lancel_2012_PLoS_One&diff=35686Lancel 2012 PLoS One2012-11-07T09:23:08Z<p>Harrison DK: </p>
<hr />
<div>{{Publication<br />
|title=Lancel S, Montaigne D, Marechal X, Marciniak C, Hassoun SM, Decoster B, Ballot C, Blazejewski C, Corseaux D, Lescure B, Motterlini R, Neviere R (2012) Carbon monoxide improves cardiac function and mitochondrial population quality in a mouse model of metabolic syndrome. PLoS One 7: e41836.<br />
|info=[http://www.ncbi.nlm.nih.gov/pubmed?term=Carbon%20monoxide%20improves%20cardiac%20function%20and%20mitochondrial%20population%20quality%20in%20a%20mouse%20model%20of%20metabolic%20syndrome. PMID: 22870253 Open Access]<br />
|authors=Lancel S, Montaigne D, Marechal X, Marciniak C, Hassoun SM, Decoster B, Ballot C, Blazejewski C, Corseaux D, Lescure B, Motterlini R, Neviere R<br />
|year=2012<br />
|journal=PLoS One<br />
|abstract=AIMS: Metabolic syndrome induces cardiac dysfunction associated with mitochondria abnormalities. As low levels of carbon monoxide (CO) may improve myocardial and mitochondrial activities, we tested whether a CO-releasing molecule (CORM-3) reverses metabolic syndrome-induced cardiac alteration through changes in mitochondrial biogenesis, dynamics and autophagy.<br />
<br />
METHODS AND RESULTS: Mice were fed with normal diet (ND) or high-fat diet (HFD) for twelve weeks. Then, mice received two intraperitoneal injections of CORM-3 (10 mg x kg(-1)), with the second one given 16 hours after the first. Contractile function in isolated hearts and mitochondrial parameters were evaluated 24 hours after the last injection. Mitochondrial population was explored by electron microscopy. Changes in mitochondrial dynamics, biogenesis and autophagy were assessed by western-blot and RT-qPCR. Left ventricular developed pressure was reduced in HFD hearts. Mitochondria from HFD hearts presented reduced membrane potential and diminished ADP-coupled respiration. CORM-3 restored both cardiac and mitochondrial functions. Size and number of mitochondria increased in the HFD hearts but not in the CORM-3-treated HFD group. CORM-3 modulated HFD-activated mitochondrial fusion and biogenesis signalling. While autophagy was not activated in the HFD group, CORM-3 increased the autophagy marker LC3-II. Finally, ''ex vivo'' experiments demonstrated that autophagy inhibition by 3-methyladenine abolished the cardioprotective effects of CORM-3.<br />
<br />
CONCLUSION: CORM-3 may modulate pathways controlling mitochondrial quality, thus leading to improvements of mitochondrial efficiency and HFD-induced cardiac dysfunction.<br />
|keywords=Metabolic syndrome, Normal diet, High-fat diet, Carbon Monoxide<br />
|mipnetlab=FR Lille Neviere R,<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k<br />
|injuries=Mitochondrial Disease; Degenerative Disease and Defect<br />
|organism=Mouse<br />
|tissues=Cardiac muscle<br />
|preparations=Permeabilized tissue, Isolated Mitochondria<br />
|couplingstates=OXPHOS<br />
|substratestates=CI<br />
|kinetics=ADP; Pi<br />
|topics=Mitochondrial Biogenesis; Mitochondrial Density, ATP; ADP; AMP; PCr<br />
}}</div>Harrison DK