Talk:Safranin

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 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

Mitochondrial membrane potential and especially

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. 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?
• 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 [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 "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 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 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)