Talk:Calcium Green: Difference between revisions

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Cell permeable Ca2+ fluorophores

See also Calcium

Question:

Can I use the cell permeable ester derivative of Calcium green-1 (Calcium Green™-1, AM, cell permeant) to measure INTRACELLULAR Ca concentrations of INTACT cells? How do I a calibrate?

Answer:

Calcium green-1 is the type of Calcium Green for the lowest Ca concentration. Since we are talking intracellular Ca Concentration calibration of Ca measurements in the chamber volume (NOT what YOU want to do!) would probable have to be performed by preparing calibration buffers --> too low concentrations for just titrating in Ca. Of course the application I always have in mind is using PERMEABILIZED cells or ISOLATED MITOCHOBDRIA and adjusting the medium in the measuring chamber to simulate the INTRACELLULAR medium. This is what we do in 95% of our applications (no matter which method: O2, TPP, pH). For Ca there is the special problem that keeping the chamber volume similar to intracellular medium usually involves buffering away all the Ca introduced by the preparation and all the chemical to protect the mitochondria. This is were the different approaches in the literature (e.g. using a low Ca buffering capacity in the initial phase of the experiment, then "out-titrating" this buffer with Ca pulses) come into play. Another application is to have INTACT cells and look at comparable high EXTRACELLULAR Ca concentrations - again looking at the Ca concentration in the bulk of the medium, NOT IN the sample. This should be rather simple due to the high Ca levels, but this Ca level will not change a lot by things happening inside the cells. In all cases so far you would use the water soluble form of Mg green (the hexapotassium salt).

However, if I understand correctly you propose to do something entirely different: I get that you want to measure INTRACELLULAR Ca concentrations in INTACT cells and this is why you bought the cell permeable form for Ca green-1 (the ester). So you would not be interested in the signal from the bulk of the medium but only from inside intact cells. While this is not what our fluorescence system is designed for, it does not mean that it would not work. Actually, if a measurement is possible at all (and I do not know if it is possible!) there are some benefits. My problem is that I know absolutely nothing about this cell permeable preparations, especially not if they are slightly water soluble and therefore how much of the fluorophore stays outside the cell. Just from looking at the Invitrogen page I gather that usually these dyes are measured when it is possible to look just at the cell(s), e.g. with fluorescence microscopy. Whether it is possible to detect the signal from inside the cell in the presence of the bulk medium around(that our sensor is also seeing) I do not know and I guess this will depend strongly on

1. The answer to the question I asked above: How much of the fluorphore is dissolved in the medium? That is how much of an (uninteresting and disturbing) background signal is generated by the medium outside?

2. Is the signal from inside the cells strong enough to be measured, considering that the detector is seeing mostly the medium outside?

The first step would be to look at the "measuring methods" applied in the literature using such cell permeable Ca-green derivatives:

Fluorescence microscopy? -not very helpful for our task
Flow cytometry: probable also not very helpful, because the measurement is still focused on a single cell at any given time
Cuvette based measurements : very helpful : If it works in a cuvette in should also work in the O2k measuring chamber.

The good point (if this works at all) is of course that you do not have to care about Ca buffering- the intact cell is taking care about that.

Calibration: You can not calibrate by either by titration or by calcium buffers because you can not set a defined Ca2+ concentration INSIDE an INTACT cell. Please refer to the manual (and the supplied references) from the fluorophores producer.

I always prefer to test a system first without any biological sample to make sure the instrument system works well. However in this case I do not know how to do this. Even if the ester derivative of Calcium green -1 has some water solubility you would still have to set up a measurement system (calibration buffers) that is rather meaningless for the proper experiment. I guess one could start by comparing the fluorescence signals of a sample without fluorophore, a sample with fluorophore and sample with fluorophore plus tons of external Ca added. Since there is no way of lowering the Ca concentration in the cell, you have to trust the literature (and Invitrogen) that the fluorophore is designed in a way that ensures that at typical intracellular Ca concentrations the chelating part of the fluorophore is partially bound to Ca2+ and partially free. You only can detect a change in signal as long as you stay away from saturation or too low binding. Whether this condition is met will also depend on the ratio fluorophore/ cells you use.

Fasching Mario 15:43, 3 January 2014 (CET)

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 __TOC__
+[[Image:BB-Bioblast.jpg|left|40px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]]
+== Popular Bioblast page ==
-== How to start a Ca2+ project ==
+:::: [[Calcium Green]] has been accessed more than
+::::*  10,000 times (2019-08-07)
+::::*  5,000 times (2016-03-27)
-A good starting point are the web pages of Invitrogen (see main page for links)and publications cited there. The first points to be addressed will be:
-* Can the problem be tackled by one of the fluorophores form Invitrogene's Ca green series? Which of them?
-* What kind of methods are published?
-What our fluorescence module actually does, is to bring the functionality of a cuvette based spectrofluormeter (for selected excitation  emission wavelengths) to the O2k chamber. So a publication using a cuvette based measurement of a Calcium green fluorophore should also be doable in the O2k with the fluorescence module. In contrast, the fluorescence module is not intended to supply the functions of e.g. a fluorescence microscope (with which e.g. it is possible to quantify the fluorescence from a single cell - we are always looking an the entire medium in the chamber, most of it will be OUTSIDE of the mitos/ cells - so typically we are monitoring concentrations outside of the sample).
-Ideally initial experiments will follow published methods but with the additional dimensions of simultaneous measurement of respiration / control of oxygen concentration. A publication that can be used as a starting point should:
-* address the problems you are interested in
-* use one of the Ca green fluorophores to do so
-* using a cuvette based approach in a (spectro)fluorometer to do so.
-With a bit more experience you may be able to follow publications that use a different Ca fluorphore by substituting it with a suitable Ca Green derivative.
-== Ca buffering ==
-Mitochondria are easily damaged even by the low calcium levels resulting from impurities in chemicals and preparations. In fact the native intracellular Ca2+ level may be below the total Ca2+ concentration in a medium introduced as impurities of standard laboratory chemicals. In respirometric experiments this is taken care of by "buffering away" all the Ca2+ with e.g. EGTA. But buffering and measuring small differences are of course at cross purpose. One strategy I have seen, is first using a very weak Ca2+ buffering, just to keep Ca2+ impurities under control and then add external Ca2+ in sufficient amounts to "out-titrate" the buffer. In the subsequent part of the experiment the mitochondria should be exposed to un-physiologicalyl high Ca2+ concentrations anyway.
 == Cell permeable Ca2+ fluorophores ==
-'''Question:'''
+::: '''See also [[Calcium]]'''
-Can I use the cell permeable ester derivative of Calcium green-1 (Calcium Green™-1, AM, cell permeant) to measure INTRACELLULAR Ca concentrations of INTACT cells? How do I a calibrate?
-'''Answer:'''
-Calcium green-1 is the type of Calcium Green for the lowest Ca  concentration. Since we are talking intracellular Ca Concentration   calibration of Ca measurements in the chamber volume (NOT what YOU want  to do!) would probable have to be performed by preparing calibration  buffers --> too low concentrations for just titrating in Ca.  Of course the application I always have in mind is using PERMEABILIZED  cells or ISOLATED MITOCHOBDRIA and adjusting the medium in the measuring  chamber to simulate the INTRACELLULAR medium. This is what we do in 95%  of our applications (no matter which method: O2, TPP, pH). For Ca there  is the special problem that keeping the chamber volume similar to  intracellular medium usually involves buffering away all the Ca  introduced by the preparation and all the chemical to protect the  mitochondria. This is were the different approaches in the literture(e.g.   using a low Ca buffering capacity in the initial phase of the  experiment, then "out-titrating" this buffer with Ca pulses) come into  play. Another application is to have INTACT cells and look at comparable  high EXTRACELLULAR Ca concentrations - again looking at the Ca  concentration in the bulk of the medium, NOT IN the sample. This should  be rather simple due to the high Ca levels, but this Ca level will not  change a lot by things happening inside the cells. In all cases so far  you would use the water soluble form of Mg green (the hexapotassium  salt).
-However, if I understand correctly you propose to do something entirely  different:  I get that you want to measure  INTRACELLULAR Ca concentrations in INTACT cells and this is why you  bought the cell permeable form for Ca green-1 (the ester). So you would  not be interested in the signal from the bulk of the medium but only  from inside intact cells. While this is not what our fluorescence system is designed for, it  does not mean that it would not work. Actually, if a measurement is  possible at all (and I do not know if it is possible!) there are some benefits. My  problem is that I know absolutely nothing about this cell permeable  preparations, especially not if they are slightly water soluble and  therefore how much of the fluorophore stays outside the cell. Just from  looking at the Invitrogen page I gather that usually these dyes are  measured when it is possible to look just at the cell(s), e.g. with  fluorescence microscopy. Whether it is possible to detect the signal  from inside the cell in the presence of the bulk medium around(that our  sensor is also seeing) I do not know and I guess this will depend  strongly on
-.) The answer to the question I asked above: How much of the fluorphore  is dissolved in the medium? That is how much of an (uninteresting and  disturbing) background signal is generated by the medium outside?
-.)Is the signal from inside the cells strong enough to be measured,  considering that the detector is seeing mostly the medium outside?
+::: '''Question:'''
+:::: Can I use the cell permeable ester derivative of Calcium green-1 (Calcium Green™-1, AM, cell permeant) to measure INTRACELLULAR Ca concentrations of INTACT cells? How do I a calibrate?
-The first step would be to look at the "measuring methods" applied in  the literature using such cell permeable Ca-green derivatives:
+::: '''Answer:'''
+:::: Calcium green-1 is the type of Calcium Green for the lowest Ca  concentration. Since we are talking intracellular Ca Concentration   calibration of Ca measurements in the chamber volume (NOT what YOU want  to do!) would probable have to be performed by preparing calibration  buffers --> too low concentrations for just titrating in Ca.  Of course the application I always have in mind is using PERMEABILIZED  cells or ISOLATED MITOCHOBDRIA and adjusting the medium in the measuring  chamber to simulate the INTRACELLULAR medium. This is what we do in 95%  of our applications (no matter which method: O2, TPP, pH). For Ca there  is the special problem that keeping the chamber volume similar to  intracellular medium usually involves buffering away all the Ca  introduced by the preparation and all the chemical to protect the  mitochondria. This is were the different approaches in the literature (e.g. using a low Ca buffering capacity in the initial phase of the  experiment, then "out-titrating" this buffer with Ca pulses) come into  play. Another application is to have INTACT cells and look at comparable  high EXTRACELLULAR Ca concentrations - again looking at the Ca  concentration in the bulk of the medium, NOT IN the sample. This should  be rather simple due to the high Ca levels, but this Ca level will not  change a lot by things happening inside the cells. In all cases so far  you would use the water soluble form of Mg green (the hexapotassium  salt).
-*Fluorescence microscopy? -not very helpful for our task
+:::: However, if I understand correctly you propose to do something entirely  different:  I get that you want to measure  INTRACELLULAR Ca concentrations in INTACT cells and this is why you  bought the cell permeable form for Ca green-1 (the ester). So you would  not be interested in the signal from the bulk of the medium but only  from inside intact cells. While this is not what our fluorescence system is designed for, it  does not mean that it would not work. Actually, if a measurement is  possible at all (and I do not know if it is possible!) there are some benefits. My  problem is that I know absolutely nothing about this cell permeable  preparations, especially not if they are slightly water soluble and  therefore how much of the fluorophore stays outside the cell. Just from  looking at the Invitrogen page I gather that usually these dyes are  measured when it is possible to look just at the cell(s), e.g. with  fluorescence microscopy. Whether it is possible to detect the signal  from inside the cell in the presence of the bulk medium around(that our  sensor is also seeing) I do not know and I guess this will depend  strongly on
-*Flow cytometry: probable also not very helpful, because the measurement is still focused on a single cell at any given time
-* Cuvette based measurements : very helpful : If it works in a cuvette in should also work in the O2k measuring chamber.
-The good point (if this works at all) is of course that you do not have  to care about Ca buffering- the intact cell is taking care about that.
+::::1. The answer to the question I asked above: How much of the fluorphore  is dissolved in the medium? That is how much of an (uninteresting and  disturbing) background signal is generated by the medium outside?
-Calibration: I am not sure that the concept of "calibration" makes any  sense at all for this application. In any way you can not calibrate by  either by titration or by calcium buffers because you can not set a  defined Ca concentration INSIDE an INTACT cell. I guess the results will  have to be qualitative.
-I always prefer to test a system first without any biological sample to  make sure the instrument system works well. However in this case I do  not know how to do this. Even if the ester derivative of Calcium green -1 has some water solubility you would still have to set up a  measurement system (calibration buffers) that is rather meaningless for  the proper experiment.    I guess one could start by comparing the fluorescence signals of a  sample without fluorophore, a sample with fluorophore and sample with  fluorophore plus tons of external Ca added. Since there is no way of lowering the Ca concentration in the cell, you  have to trust the literature (and Ivitrogen) that the fluorophore is  designed in a way that ensures that at typical intracellular Ca  concentrations the chelating part of the fluorophore is partially bound  to Ca and partially free. You only can detect a change in signal as  long as you stay away from saturation or too low binding. Whether this  condition is met will also depend on the ration fluorophore/ cells you  use.
+::::2. Is the signal from inside the cells strong enough to be measured,  considering that the detector is seeing mostly the medium outside?
-best greetings
+:::: The first step would be to look at the "measuring methods" applied in  the literature using such cell permeable Ca-green derivatives:
-Mario
-[[User:Fasching Mario|Fasching Mario]] 15:43, 3 January 2014 (CET)
+::::* Fluorescence microscopy? -not very helpful for our task
+::::* Flow cytometry: probable also not very helpful, because the measurement is still focused on a single cell at any given time
+::::* Cuvette based measurements : very helpful : If it works in a cuvette in should also work in the O2k measuring chamber.
-== Ca calibration ==
+:::: The good point (if this works at all) is of course that you do not have  to care about Ca buffering- the intact cell is taking care about that.
+:::: Calibration: You can not calibrate by  either by titration or by calcium buffers because you can not set a  defined Ca2+ concentration INSIDE an INTACT cell. Please refer to the manual (and the supplied references) from the fluorophores producer.
-Question: Which Ca buffers should I prepare to calibrate Ca measurements via fluoresecence / or via ca electrode?
+:::: I always prefer to test a system first without any biological sample to  make sure the instrument system works well. However in this case I do  not know how to do this. Even if the ester derivative of Calcium green -1 has some water solubility you would still have to set up a  measurement system (calibration buffers) that is rather meaningless for  the proper experiment.    I guess one could start by comparing the fluorescence signals of a  sample without fluorophore, a sample with fluorophore and sample with  fluorophore plus tons of external Ca added. Since there is no way of lowering the Ca concentration in the cell, you  have to trust the literature (and Invitrogen) that the fluorophore is  designed in a way that ensures that at typical intracellular Ca  concentrations the chelating part of the fluorophore is partially bound  to Ca2+ and partially free. You only can detect a change in signal as  long as you stay away from saturation or too low binding. Whether this  condition is met will also depend on the ratio fluorophore/ cells you  use.
-Answer:  Unfortunately it makes no sense  to recommend any calibration procedure or calibration buffers before the user has some idea about the project she/he wants to do. First of all,       which Ca concentrations does she want to measure? Eg. The "Tsien" series of buffers I used in the development of the Ca electrode, covers Ca concentrations between 10E-3 to 10E-8 M. No biologist will need the entire range. Maybe the user wants to measure extracellular Ca levels? - then no       buffered calibration solution will be necessary at all. Then the question is which method, fluorescence or Ca electrode should be addressed. For this question the technical experience of the customer will be a major deciding factor (more technical experience necessary for the Ca electrode than for fluorescence). Would it be possible to tackle the particular question  with any Ca       green fluorophore?  If the user goes for fluorescence there is quite a lot of  application information available on the Invitrogen website.  But even in this case the user WILL have to spend some time for method development / establishing the method.
+:::: [[User:Fasching Mario|Fasching Mario]] 15:43, 3 January 2014 (CET)