Ray, This sound like two different experiments - can he split the population after transfection and look at the viability of one half with Calcein-AM / PI and fix the other half to do the intracellular antibody staining? If you want to try someting along the lines of what you described, it would be better to use a higher-affinity DNA intercalator like ethidium homodimer so there would be less transfer of dye from the previously dead cells to cells that were once alive but are dead after fixation.. Best regards, Michael Kuhn, President Helix Research Fluorescence Chemistry mkuhn@helixresearch.com www.helixresearch.com ----- Original Message ----- From: ray hester <rhester@jaguar1.usouthal.edu> To: cyto-inbox Sent: Friday, August 31, 2001 12:22 PM 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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