Dear Flowers.... € small point about JC-1 and apoptosis in answer to: >>I'm using JC-1 to look at the time couse of mitochondrial specific events >>in drug-induced apoptosis. Basically, I treat my cells with drug for >>various time points (Up to 24hrs maximum), after which I harvest adherent >>and non-adherent cells and stain for 15 min at 37 degrees in 1uM of JC-1. >>Instead of depolarisation (a change from orange aggregate formation to >>green fluorescence), I see a hyperpolarization ....my drug treated cells >>are bright orange (even more than controls), reaching a maximum intensity >>at the 24 hr time point. These cells I know to be 80 - 90 % apoptotic, and >>I can even see a classic apoptotic morphology (blebbed membranes) with the >>bright orange mitochondrial staining. >>Since the drug I'm using is very hydrophobic could this be having an effect >>on the mitochondrial membrane potential alone...or could it somehow be >>accumulating in mitochondria? >>Any suggestions would be gratefully appreciated. >>Hayley McDaid, Ph.D. >>Molecular Pharmacology >>Golding Room 201 >>Albert Einstein College of Medicine >>1300 Morris Park Avenue >>Bronx, New York 10461 >> >>Telephone: 718-430-2192 >>Fax: 718-430-8922 >>E-mail: mcdaid@aecom.yu.edu Dear Hayley, The facts you described here are one of the numerous problem entountered with the use of JC-1 probe in the scope of apoptosis. It is clear that flow cytometry cannot solved all problem and that any study has to be accompagnied with biochemical, image cytometry and eventually electron microscopy. The different points are: € 1 € Clearly yo can lower the JC-1 concentration to 1 nM. Taking care that your stock solution do no contain aggregate. That is the basic point... because of the well known effects of the probes onto the mitochondrial metabolism. € 2 € The second point is... and I would like to be cited as personnal communication.... That during the course of apoptosis, many cells exhibit an intense vacuolization... as part of a developed detoxification network.... And we have clearly observed that apoptotic cells with a depolarized mitochondrial compartment... have a high orange fluorescence (which only transiently appears)... linked to a high staining of the vacuolar system with JC-1 (staining which of course was not present at start). As a consequence of this is not the mitochondrial membrane potential which has to be considered as responsible of the JC-1 fluorescence in the developing apoptosis but the capability of the developing vacuolar system to take the probe. € 3 € A solution is available... we have submitted an article... Please contact me directly Sincerely yours Patrice PETIT \ | / (o o) \ | / _______________oOo_____oOo_____oOo_ (o o)__oOo________________ Dr. Petit Patrice X. Institut Cochin de Génétique Moléculaire INSERM U129 - CHU Cochin Port-Royal 24, rue du Faubourg Saint-Jacques F-75014 Paris, France. Tel: 33 01 44 41 24 11 Fax: 33 01 44 41 24 21 E-mail: pxpetit@icgm.cochin.inserm.fr
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