I can't quite understand the discription of your problem, but when I want to look at FITC and PI at the same time, I usually read PI at 675nM (FL3 on a BD Facscan). Then I don't have to fool with the compensation settings. Sincerely, Dan Rosson Dan Rosson Ph.D. Lankenau Institute of Medical Research 100 Lancaster Ave. Wynnewood, PA 19096 www.limr.org. -----Original Message----- From: Silvi Rouskin [mailto:srouskin@fit.edu] Sent: Saturday, June 08, 2002 3:24 PM To: cyto-inbox Subject: FITC and PI compensation Hi, I am analyzing cyclins in mouse leukemia cells, and I am staining with FITC and PI but I am not quite sure how to do compensation. So far I have used single stained samples and tried to make the median fluorescence in the other channel equal to the median fluorescence of the unstained cells for that channel by moving the compensation settings. I ran into a few problems. First, in some experiments, the FITC stained cyclins had less fluorescence in FL2 than the unstained. Second, when I figure out compensation settings, should I read my control (unstained cells or IgG, with PI) in the same compensation settings as the cycins? Because, if I compensate the control , it gets much lower in FL1 than the cyclin and I cannot use it as a region to quantitative the cyclin expression. I would really appreciate it if someone could give me any ideas or information on how to compensate properly with the cyclins and the control. Thank you very much! Silvia Rouskin Undergraduate cancer research Florida Institute of Technology
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