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Talk:Safranin

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TPP vs safranin


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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:

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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