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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