This is a copy of the letter posted by Prof. Michael Duchen to MITOCHONDRIA mail list in 2007. It might be helpful to someone thinking of using JC-1 dye to measure the mitochondrial membrane potential.

    JC-1 has been a thorny issue for years. We used it as soon as it became available in the early 90's and published a paper or two. However we soon found that for dynamic measurements of potential it is very, very slow to change compared with TMRM or a TPP⁺ electrode. Imaging JC-1 signals generates some strange images. Loading for short periods and then washing, which is the usual way the dye is used, always seems to show peripheral mitochondria stained red and central mitochondria green (i.e. there is more dye in the peripheral mitochondria and so formation of J aggregates). Many mitochondria also look strange - they tend to show elongated spindle like structures that are never ever seen if mitochondria are labelled with mtGFP or TMRM or mitotrackers. The fact that peripheral mitochondria are often stained red has been interpreted to mean that these mitochondria have a bigger potential. However, if you load with very low dye concentrations and leave for a long time (several hours) to equilibrate, all the mitochondria are uniformly stained. This is consistent with the proposal that JC-1 is very slow to cross membranes and therefore very slow to equilibrate, as discussed by David Nicholls in several publications.

There are also published data from Christos Chinopoulos to suggest that the red signal is influenced by ROS.

My hunch is that the dye might be useful but that no-one has invested the necessary time to establish ideal conditions to use it to give reliable data. It seems too slow to be useful to follow dynamic changes in potential but it may still prove useful to compare potential between populations if used correctly.