Difference between revisions of "Talk:Safranin"

Latest revision as of 15:14, 7 August 2019

TPP vs safranin

MitoPedia O2k and high-resolution respirometry: O2k-Open Support

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:

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

It must be established that the relationship is linear (or at least known) for the experimental conditions.
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 already established the safranin technique method using spectrofluormetry, subsequently to obtain a TPP electrode as well.

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.

Summarising, the differences between the safranin and TPP methods are:

If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.
If quantification of differences is sufficient then safranin and TPP are both suitable.
The type of sample may make one method or the other more suitable.
The decision also relies on the types of calibration methods that may be required.
There is probably always a benefit of having both methods available.
Inhibition by safranin is a problem to be considered with all cations used to measure mtMP.

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

Answer: We have to discern between the SUIT protocol protocol for the addition of substrates, uncouplers, etc, and additional steps necessary for safranin or TPP. Neither safranin nor TPP require major modifications to the SUIT protocol, apart from excluding incompatible chemicals. Both methods can be used with a wide range of different SUIT protocols.

--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 safranin 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:

With DatLab 6 an Excel spreadsheet is no longer required: »MiPNet19.19 DatLab 6
Krumschnabel 2014 Methods Enzymol
MiPNet20.13 Safranin_mt-membranepotential
Figueira 2012 Methods Mol Biol

In many publications, e.g. Komary 2010 Biochim Biophys Acta, no calibration was done and only the plot of the fluorescence intensities is presented. A simple two point or multiple point calibration for safranin concentration is recommended, and a correction for chemical background effects. A claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. When safranin is titrated in the presence of sample, 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. Fasching Mario 09:13, 10 January 2013 (CET)

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@@ Line 1: / Line 1: @@
 __TOC__
-==TPP vs Safranin==
+== TPP vs safranin ==
+{{Technical support integrated}}
 '''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 Instruments:'''
-We obviously have more experience with TPP than with safranin.
-One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", please see
+'''Answer by OROBOROS:'''
+One of the big issues with measuring mitochondrial membrane potential is how to get "absolute values", see
 [[Mitochondrial membrane potential]]
@@ Line 16: / Line 16: @@
 [[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.
-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
+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:
-* some calibration is necessary, even for an established linear relationship
+* It must be established that the relationship is linear (or at least known) for the experimental conditions.
+* 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.
+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.
-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.
+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.
-Therefore important considerations re safranin / TPP are:
+Summarising, the differences between the safranin and TPP methods are:
-* are absolute values required?
+* If absolute measurements of membrane potential in isolated mitochondria are required, TPP is the method of choice.
-* is at least a quantification of differences (absolute differences) required?
+* If quantification of differences is sufficient then safranin and TPP are both suitable.
-* what types of sample are of interest?
+* The type of sample may make one method or the other more suitable.
-* what calibration methods  are available
+* The decision also relies on the types of calibration methods that may be required.
-* if possible, there is probable always a benefit of having both methods available
+* There is probably always a benefit of having both methods available.
+* Inhibition by safranin is a problem to be considered with all cations used to measure mtMP.
-'''More input from users to this important topic is required! Please contact  [email protected] 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 [[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.
+'''Answer:''' We have to discern between the [[Substrate-uncoupler-inhibitor titration|SUIT  protocol]] protocol for the addition of substrates, uncouplers, etc, and additional steps necessary for safranin or TPP. Neither safranin nor TPP require major modifications to the SUIT protocol, apart from excluding incompatible chemicals. Both methods can be used with a wide range of different SUIT protocols.
-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:
-# deciding on a safranin concentration and  sample to safranin ratio
-# selection of excitation and emission wavelength and bandwidth
-# addition of safranin
-# addition of sample and start of desired SUIT protocol
-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 [[O2k-Fluorescence_LED2-Module#Application_specific_settings|application specific settings]].
-For an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].
 --[[User:Fasching Mario|Fasching Mario]] 12:32, 4 May 2012 (CEST)
-== Safranin Calibration ==
+== 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?
+I now would like to use safranin 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:'''
-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]].
+* With DatLab 6 an Excel spreadsheet is no longer required: »[[MiPNet19.19 DatLab 6]]
+* [[Krumschnabel 2014 Methods Enzymol]]
+* [[MiPNet20.13 Safranin_mt-membranepotential]]
+* [[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. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)
+In many publications, e.g. [[Komary 2010 Biochim Biophys Acta]], no calibration was done and only the plot of the fluorescence  intensities is presented. A simple two point or multiple point calibration for safranin concentration is recommended, and a correction for chemical background effects. A claimed linear relationship with the mitochondrial membrane potential is based on the safranin fluorescence signal and not the actual safranin concentration. When safranin is titrated in the presence of sample, 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. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)
-== Inhibition by safranin ==
-Safranin may inhibit mitochondrial function. 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)
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