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Difference between revisions of "Talk:Safranin"

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[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]
[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]


For the TPP method absolute values can be obtained for isolated mitochondria 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.
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.
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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:
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:
* It must be established that the relationship is linear (or at least known) for the experimental condition.
* It must be established that the relationship is linear (or at least known) for the experimental condition.

Revision as of 13:43, 2 June 2015

TPP vs safranin

Question: 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?


Answer by OROBOROS: Update: please see also: MiPNet19.19 Safranin Data Acquisition and Analysis and Krumschnabel 2014 Methods Enzymol.

One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see

Mitochondrial membrane potential and especially

Calculation of mitochondrial membrane potential from measurements with a TPP electrode

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.

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:

  • It must be established that the relationship is linear (or at least known) for the experimental condition.
  • Some calibration is necessary, even for an established linear relationship.

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.

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.

Therefore important considerations re safranin / TPP are:

  • Are absolute values required?
  • Is at least a quantification of differences (absolute differences) required?
  • What types of sample are of interest?
  • Which calibration methods are available?
  • There is probably always a benefit of having both methods available.

Inhibition by safranin may also be a major disadvantage of the safranin method.


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!

Follow up question: Whats the difference between a TPP and a safranin protocol?

Answer: We have to discern between the protocol for the addition of substrates, uncouplers, etc, lets call it "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. 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:

  1. Deciding on a safranin concentration and sample to safranin ratio.
  2. Selection of excitation and emission wavelength and bandwidth.
  3. Addition of safranin.
  4. Addition of sample and start of SUIT protocol.

Update: please see also: MiPNet19.19 Safranin Data Acquisition and Analysis and Krumschnabel 2014 Methods Enzymol.

Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by an other method is not done in the same experiment.


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 application specific settings.

For an example of a protocol used together with safranin see Komary 2010 Biochim Biophys Acta.


--Fasching Mario 12:32, 4 May 2012 (CEST)

Safranin calibration

Question: 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! 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?

Answer:

Update: please see also: MiPNet19.19 Safranin Data Acquisition and Analysis and Krumschnabel 2014 Methods Enzymol. 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.

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. Fasching Mario 09:13, 10 January 2013 (CET)

Inhibition by safranin

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! Fasching Mario 09:12, 10 January 2013 (CET)

This problem seems to be quite severe. Laner Verena 14:36, 16 April 2013 (CEST)

Safranin chemical background

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. Fasching Mario 09:19, 10 January 2013 (CET)


Substances with an effect on the fluorescence signal of safranin

  • ADP (D)
  • Cytochrome c (c)
  • Succinate (S)
  • Rotenone (Rot)
  • Ascorbate (As)
  • TMPD (Tm)


Substances without an effect on the fluorescence signal of safranin

  • Pyruvate (P)
  • Malate (M)
  • Glutamate (G)
  • Digitonin (Dig)
  • Oligomycin (Omy)
  • FCCP (U)
  • Malonic acid (Mna)
  • Antimycin A (Ama)
  • DMSO
  • Ethanol
  • H2O2
  • H2O