JC-1

James Marvin jmarvin at flowcity.bsd.uchicago.edu
Thu Jun 17 17:12:19 EST 2004


Hello,

I was wondering if those of you who have use JC-1 would be willing to share 
some info with the rest of us.
I have come across some interesting data in the literature pertaining to 
this dye.  Chinopoulos et al,  for one have shown that the decrease in 
aggregate (590nm, FL-2, PE detector, 585/30, or whatever you call it at 
your institution) fluorescence in response to H2O2 (a common control for 
oxidative stress experiments, I think) is not dependant on mitochondrial 
membrane potential.  After stimulation with FCCP they showed a further 
decrease in aggregrates after H2O2 production.	I believe that the exact 
reason for the fluorescence decrease in response to H2O2 is undetermined. 
Di Lisa et al have suggested that the decrease in the aggregate emission 
derives not only from the release of the probe from the mitochondrial 
matrix to the cytosolic space but also form the interaction of JC-1 with 
negative charges of the inner mitochondiral membrane.  Increase in 
monomeric form of JC-1 seems to correlate with membrane potential, but this 
is also probably a complex measurement that might not always be accurate.

Sometimes it seems that interpretation of some flow data can be 
oversimplified.  ROS detection and mitochondrial membrane potential in 
particular.


Anyway, I was just wondering what types of positive and negative controls 
people use with this dye. And what gates people use to analyze their 
data.  Do you incorporate everything that shifts from your normal sample, 
in any direction?  What type of preliminary work do you do in order to set 
up proper staining conditions?	(This dye seems to be extremely 
concentration dependant).  What are some variables that effect/alter Nernst 
determined dye loading.  I work in a core facility and see lots of 
different cell types and assays.  There doesn't seem to be any consistency 
between different cell types, controls etc.  To be perfectly honest I 
really don't like this dye.  I have had much better luck with the 
mitotrackers and DIOC's.  But I can't seem to get people to stop using it 
so I might as well understand it a little better.

Any info or references are welcome
Have a great day
Marv

1.	   Chinopoulos, C., L. Tretter, and V. Adam-Vizi, Depolarization of 
In Situ Mitochondria Due to hydrogen peroxide-induced oxidative stress in 
nerve terminals: inhibition of a-ketoglutarate dehydrogenase. Jounal of 
Neurochemistry, 1999. 73: p. 220-228.

19.	  Di Lisa, et al., Mitochondrial membrane potential in single 
living adult rat cardiac myocytes exposed to anoxia or metabolic 
inhibition. J. physiol, 1995. 486: p. 1-13.




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