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: Update: please see also: MiPNet19.19 Safranin Data Acquisition and Analysis and Krumschnabel 2014 Methods Enzymol.

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

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 may also be a major disadvantage of the safranin method.

More input from users to this important topic would be welcome. 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, referred to here as the "SUIT protocol" and specific additional steps necessary for safranin or TPP, referred to as the "specific protocol". Unlike other methods neither safranin nor TPP require major modifications to the "SUIT protocol", apart from excluding incompatible chemicals. That is to say, 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 in addition to the SUIT protocol. The "specific protocol" for safranin is quite straighforward. In the normal case of using it in a spectrofluorometer cuvette, this would include:

Deciding on a safranin concentration and sample-to-safranin concentration ratio.
Selection of excitation and emission wavelength and bandwidth.
Addition of safranin.
Addition of sample and start of SUIT protocol.

Update: please see also: MiPNet19.19 Safranin Data Acquisition and Analysis and Krumschnabel 2014 Methods Enzymol.

Calibration of the safranin fluorescence by comparing the results with membrane potentials obtained by another method should be done in a separate experiment.

If using 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)

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?

Answer:

Update: please see also: MiPNet19.19 Safranin Data Acquisition and Analysis and Krumschnabel 2014 Methods Enzymol. That depends on whether quantification is required and if so what kind. Safranin is mainly a qualitative method, however for isolated mitochondria the literature claims that for certain safranin to sample concentration 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.

For other samples (e.g. permeabilised cells) this relation, as shown in the literature, is definitely non-linear. In the first instance, it is advisable to consult the literature published by workers using safranin in cuvettes to see what calibration procedures they used, and at what point they introduced the safranin. However, in many papers, e.g. Komary 2010 Biochim Biophys Acta, no calibration was done and only the plot of the fluorescence intensities is presented. This is an honest, pragmatic approach. Nonetheless, independent of your final format for publication, as a minimum, a simply two point calibration for safranin concentration is recommended. 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 quantification is required, a multiple point calibration could be used to obtain more precise safranin concentrations. However the claimed linear relationship with the mitochondrial membrane potential is 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 should the safranin be introduced into the chamber for calibration purposes? If you inject the safranin for the calibration before the sample it will be easy to set the markers. However, the very different absorption properties of the solution without the sample compared to that with the sample means that sensitivity will probably be quite different. When the safranin is injected after the sample, the sensitivity will be correct but 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. However, If full quantification is not required, any small error here should be negligible. If the display of fluorescence intensity in a plot together with plots from two different sensors is desired, this (displaying roughly calibrated safranin concentrations) could be a recommended approach, otherwise there will be an 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 suitable, maybe labelling the y axis of the resulting plot "arbitrary units" which is possible in the newest release of DatLab 5. Fasching Mario 09:13, 10 January 2013 (CET)

Inhibition by safranin

Safranin inhibits mitochondrial function (Krumschnabel 2014 Methods Enzymol). Always check the influence of the safranin concentration used on the respiratory rate. Insufficient data is available to define "safe" safranin concentrations for different sample types. Please add your own experiences here. Fasching Mario 09:12, 10 January 2013 (CET)

This problem seems to be quite severe. Laner Verena 14:36, 16 April 2013 (CEST)

Safranin chemical background

Several substances typically used in SUIT protocols may influence the fluorescence signal from safranin when injected into the O2k-Chamber. The chemical used should be tested for this effect in a background run without a biological sample. If necessary corrections should be applied. Strongly colored substances such as cytochrome c can be expected to have such an effect. A significant effect has also been found with ADP. Fasching Mario 09:19, 10 January 2013 (CET)

Substances with an effect on the fluorescence signal of safranin

ADP (D)
Cytochrome c (c)
Succinate (S)
Rotenone (Rot)
Ascorbate (As)
TMPD (Tm)

Substances without an effect on the fluorescence signal of safranin

Pyruvate (P)
Malate (M)
Glutamate (G)
Digitonin (Dig)
Oligomycin (Omy)
FCCP (U)
Malonic acid (Mna)
Antimycin A (Ama)
DMSO
Ethanol
H₂O₂
H₂O