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. Professor Michael Duchen about JC1:
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.
We also found that in response to several reagents the JC-1 signal
changed in ways that were not consistent with basic bioeneregtic
principles and we therefore abandoned the dye.
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.
I hope that clarifies our experience with this dye, and I believe that
this is an experience shared with several other labs.
With best regards
Michael
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