Ray, If the intent is to determine the number of cells dead before the permeabilization process, then use ethidium monoazide (EMA) from Molecular Probes. It only enters dead cells, is fixed in place with a 10 minute blast of light, and then the excess can be washed out before the permeabilization. Dead cells are found in the FL3 channel (red, same as PI). It is highly compatible with FITC. Randy T. Fischer NIH/NIAMS Building 10, Room 6D57 9000 Rockville Pike Bethesda, MD 20892 (301) 594-3537 fischer1@mail.nih.gov > ---------- > From: ray hester > Sent: Friday, August 31, 2001 3:22 PM > To: Cytometry Mailing List > Subject: dead cell marker - post fixation > > > Hi, > > An investigator wants to identify transfected cells with an > FITC-conjugated > antibody to an intracellular marker (so the cells will have to be fixed > and > permeabilized) and at the same time he wishes to know the percentage of > these transfected cells that are viable/non-viable. > > I assume that propidium iodide is out, since all of the cells will > fluoresce > with this dye after fixation and permeablization, however > > I also seem to remember that propidium iodide isn't covalently bound to > DNA > and that it will diffuse out over time if the PI-stained cells aren't kept > in PI-buffer, but is it possible to do the transfection, add the PI (I'm > not > sure what the time frame is here), fix and permeablize the cells, stain > with > the anti intracellular marker antibody, and do a flow analysis before > substantial amounts of the PI have left the > dead-as-a-result-of-transfection > cells (the anti intracellular marker antibody doesn't have to be FITC > conjugated, I don't believe, if that helps at all)? > > If the answer to this is, no, are there any dead-cell markers/antigens to > which antibodies are available? > > Thanks. > > Ray Hester > Univ. of South Alabama > rhester@jaguar1.usouthal.edu > > >
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