I am posting this on behalf of the person below. Please respond directly to tim_oneill@merck.com - Thanks Paul ------- Forwarded Message Follows ------- Tim O'Neill (tim_oneill@merck.com) Help! PI Staining: Events/second problem Hello all, I have been doing standard FACScan PI analysis for DNA content using cultured rat fibroblasts and a human carcinoma line. My problem is that when I go to analyze, my sample settles in the tube extremely easily and I constantly have to take off the tube, resuspend, and keep collecting until I get 10-15,000 events, which takes forever! The last time I did this, I could barely collect 2000 events. Is it that I just did not seed a high enough concentration of cells? Regardless of what my cell count is when I harvest, I resuspend or concentrate to 2 million/ml. My basic procedure is as follows: trypsinize, resuspend in media, pellet at low speed (1200rpm), resuspend at 2 million/ml in PBS, add equal volume of 70% ethanol overnight or longer, pellet cells at low speed (1200rpm), resuspend in PBS, aliquot 1.0 ml of this to a flow tube, spin low, resuspend back up in 1.0ml of PI staining solution from Becton (50ug/ml), add RNAse (100ul of 0.5mg/ml), incubate at 37C for 30 min, set on ice until ready to analyze. I haven't done any vortexing. Should I be? Or does this sound like an instrument problem to anyone? Is there anything obvious here that I am missing or doing wrong? I do get pretty good histograms once I collect. Thanks in advance for anyone's help. Tim Merck Research Labs (tim_oneill@merck.com) ------------------------------------------- Posted by J.Paul Robinson, Purdue University Cytometry Labs Professor of Immunopharmacology robinson@flowcyt.cyto.purdue.edu PH:765-494 6449 FAX:765-494 0517 web http://www.cyto.purdue.edu
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