The alteration in mitochondrial transmembrane potential (Delta sigh m/DWm font mismatch here) can be determined using the 5,50,6,60-tetrachloro-1,10,3,30-tetraethyl benzimidazolyl carbocyanine iodide (JC-1 dye) which is commonly used to detect mitochondrial depolarization that occurs during the early stages of apoptosis.

In healthy cells, JC-1 accumulates in the mitochondria as JC-1 aggregates (fluorescence of which is red) and also in the cytoplasm as JC-1 monomers (fluorescence of which is green). During early apoptosis, the DWm collapses. Consequently, JC-1 aggregates do not accumulate within the mitochondria and so dissipate as JC-1 monomers leading to loss of red fluorescence. Therefore, collapse of the (DWm) is indicated by decrease in the ratio of red to green fluorescence.

JC1 dye, in practice, is highly unreliable dye. At the best, it gives only qualitative comparative (with some other cells) evaluation. In situ, quantitative evaluation of the mitochondrial membrane potential (MP) is very difficult, if possible et all. With the isolated mitochondria, it is possible quantify MP with the hydrophobic cations using easily made electrodes. There are, though, a number of "subtle pitfalls" and limitations, which impossible to describe here. I have summarized these in my book "Practical Mitochondriology", which you can find on Amazon.com (self published

Indeed, results are very cell line dependent. After induction of apoptosis, the green flourescence should go up, and the orange down when measured by FCM (see this website: http://www.meduniwien.ac.at/user/johannes.schmid/JC1staining.htm for a good protocol), but I've seen certain cell lines do the opposite, or results were not quite concordant between FCM and microscopy. It may help to reduce the amount of dye used. Allways include positive and negative controls when you use this technique.

You can also use DiOC6(3) or TMRM, both of which require some tuning and positive (CCCP-treated cells) and negative (healthy cells) controls. If I don't mix things up, TMRM is compatible with fixation and fluorescence microscopy. DiOC6 (3) surely isn't.

You may want to read PMID 19373242, 18662579 and 17294081

it is better to start with DIOC6(3) using CCCP and Valinomycin as positive controls then shift to JC1. best of luck

We already had a simillar discussion:

https://www.researchgate.net/post/What_is_the_best_probe_to_evaluate_change_in_membrane_potential_JC1_or_TMRE2

Maybe it can help.

A relatively simple way is to use TMRE or TMRM and confocal microscopy. Image at 12-bit or higher so you have at least 4096 steps of gradient (vs 256 with 8-bit). Use fluorescence intensity of mitochondria and cytosol (or nucleus as substitute for cytosol if your cells are full of mitochondria) to calculate millivolt delta psi mt.

See pg 2 to 6 of published protocol by Lemasters and Ramshesh, Methods in Cell Biology: http://academicdepartments.musc.edu/ccdir/ramshesh.pdf

I guess there are many dye and technique available for measuring mitochondrial membrane potential.

1. JC1 and JC10 , I guess selection depend upon techniques and cell types. For example JC1 may be used in flow cytometry while JC10 for microplate assay.

The best way is to use a TMRM or a TMRE kit with CCCP-treated cells as positive control