Hello everyone, Sraboni here. I'm new to the mailing list and a graduate student. I'd be grateful for any experience/thoughts/ideas on the following: I'm using a FACScan to do a promoter readout assay with GFP, which I transfect by electroporation in a megakaryocytic cell line. As an intrinsic readout for rate of transfection my electroporation medium has 5. 10 -6 M propidium iodide, the reasoning being that in a population gated for live cells only those that reseal their membranes after the electroshock will be PI positive. The problem is that in the condition where cells haven't been tansfected but still kept in PI overnight, the live cell population has about 1% PI positive cells. The Molecular Probes catalogue says that PI is intact membrane impermeable, so live cells should not be stained at all. If, as someone here says, these cells are necrotic, shouldn't they become smaller than viable cells and move to the left on the FCS axis? My question is: why are some live cells staining positive for PI? Could anyone suggest another marker I could use as an intrinsic transfection indicator which could be excited by an argon laser? Thanks for your help. Sraboni ____________________________________________________________________ Ms Sraboni Ghose Tel: 41 31 632 2526 Pharmakologisches Institut 41 31 632 3281 Universitaet Bern Fax: 41 31 632 4992 Friedbuehlstrasse 49 CH-3010 Bern Switzerland Studentenlogierhaus Fellergut Tel: 41 31 992 8064 Muehledorfstrasse 28 CH-3018 Bern Switzerland Life is what happens to you while you're busy making other plans.
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