From: Caleb Lee (caleb.lee@tufts.edu)
Date: Wed Sep 03 2003 - 08:33:56 EST
Hello. I'm a graduate student in Naomi Rosenberg's lab at Tufts. In my experiment, I incorporate brdU into cell lines and then gamma irradiate the cells. After washing out the brdU, I wait about 12 hours, fix and stain for brdU using the kit by BD Pharmingen (uses paraformaldehyde and DNase I), and then stain for cell cycle analysis using PI. The irradiated cells should be either apoptotic or G2 arrested. I'm hoping to detect a difference in the irradiation response in G1 versus S phase cells as shown by gating for brdU incorporation. In healthy populations without gamma irradiation this works fine. With gamma irradiation, it still works fine if I only look at brdU staining or PI staining alone. The problem is that when I combine all three, the PI staining is no longer consistent. The patterns of the histograms are ok; I can see apoptotic and G2 arrested cells. But the intensity of the G2 peak varies. It can be either lower or higher than expected based on the location of the peak in unirradiated samples. Can someone let me know a possible reason for this and any way to fix it/get around it? Thanks a lot. Caleb
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