David, Have you tried any of the MitoTracker dyes from Molecular Probes instead of JC-1? Eric >Greetings, > > I have an investigator who sent the note below to me after we >together did >the FACS and fl. microscope observation...... > The cells treated with valinomycin were nicely depolarized and showed a >stong green (little orange) signal. The cells in question were bright >orange and indeed full of bright orange mitochondrion. > > Together we are stumped...hence the note to , as we say here in Da >Bronx.....Da List! > >> >>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 >> >> >> >> >Dave Gebhard--Director FACS Facility >Albert Einstein College of Medicine >Chanin Building 309 >1300 Morris Park Avenue >Bronx, New York 10461 >718-430-2724/ 3573 FAX 718-430-8550 /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
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