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)

Popular Bioblast page

Safranin has been accessed more than

10,000 times (2019-08-07)
5,000 times (2016-02-27)

@@ Line 1: / Line 1: @@
-==TPP vs Safranin==
+__TOC__
+== 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 14: / 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
-* 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.
+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.
-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.
+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.
-Therefore important considerations re safranin / TPP are:
+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.
-* are absolute values required?
+Summarising, the differences between the safranin and TPP methods are:
-* is at least a quantification of differences (absolute differences) required?
-* what types of sample are of interest?
-* what calibration methods  are available
-* if possible, there is probable always a benefit of having both methods available
-'''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'''!
+* 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 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 incompatibel 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.
+'''Follow up question''': Whats the difference between a TPP and a safranin protocol?
-There is no "specific protocol" for safranin that I am am aware of beside - obviously - adding safranin, though maybe one could call the various preferred  safranin / sample ratios such a "protocoll". 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 an example of a protocol used together with safranin see [[Komary 2010 Biochim Biophys Acta]].
+'''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.
 --[[User:Fasching Mario|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. [[User:Fasching Mario|Fasching Mario]] 09:13, 10 January 2013 (CET)
+[[Image:BB-Bioblast.jpg|left|40px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]]
+== Popular Bioblast page ==
+[[Safranin]] has been accessed more than
+::::*  10,000 times (2019-08-07)
+::::*  5,000 times (2016-02-27)