Dear Colleagues: I have been doing cell cycle analysis and if you could help me with following questions, I would be very much appreciative. The system I have is to co-transfect HeLa cells with transfection marker (GFP-F) and a gene of my interest on transient mammalian expression vector (pFLAG) and harvest them at 16 hours, fix them with 70% MetOH at -20C for 2 hours and stain with PI and do flow cytometry. Recently, I have been getting clear difference in cell cycle distribution patterns by eyes between control vector (pFLAG only) and my gene. However, when I use ModLit program, the difference is not as impressive. I am using Manual Fit: Frozen, Diploid, No aggregate, Visible G2, No apoptosis. I gate the cells for GFP-F and I remove aggragates using Am I doing something wrong? Thank you very much for your time and help.
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