JC-1 Dioc6

Craig.Turner@nbs.nhs.uk Craig.Turner at nbs.nhs.uk
Mon Sep 4 03:57:34 EDT 2006


Howard your comments on JC-1 are interesting. There are groups in the
platelet field reporting JC-1 ratio for mitochondrial potential and
currently in our lab a colleague is working with a method based on the
published work. His major problem seems to be getting consistent
fluorescence between batches of dye and I don't think he has nailed down a
storage condition he is entirely happy with. All of which means the ratio
becomes unreliable as a comparison of mitochondrial potential over time.
Does anyone else have this sort of problem or any hints I can pass on to
improve the process?
Craig 
Craig Turner PhD 
Scientific development supervisor 
Components Development Laboratory 
NBS Brentwood 


-----Original Message-----
From: Howard Shapiro [mailto:hms at shapirolab.com]
Sent: 31 August 2006 01:38
To: cyto-inbox
Subject: Re: JC-1 Dioc6



Paul Waring wrote:


I have been out of touch with the mitochondria field for a few years. Would
anyone care to give me an update on the relative advantages of JC-1 versus
Dioc6 (or other dyes) in measuring mitochondrial membrane potential ?


JC-1 has been found useful for discriminating energized from deenergized
mitochondria, but, as far as I know, has never been demonstrated to
*measure* mitochondrial potential. The distribution of green vs. red
fluorescence is too broad in all directions to make a ratiometric procedure
feasible. 


It is claimed that DiOC6(3) at very low concentrations (around 1 nM) *can*
be used for quantitative measurements (Rottenberg H, Wu S: Quantitative
assay by flow cytometry of the mitochondrial membrane potential in intact
cells. Biochim Biophys Acta 1404:393-404, 1998). I suspect that other
cyanines would also work at this low concentration. If the mitochondria are
energized,  the dye concentration inside will be at least 100 times the
external concentration; if the external concentration is much higher than 1
nM, there will be substantial quenching of intramitochondrial dye
fluorescence, and also more interference from dye in the cytosol. At the
lower concentrations, mitochondrial fluorescence would be expected to track
membrane potential.


-Howard




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