Flowers, I've had time to go back and look at this data again. I've included my comp controls and what to (my) naked eye looks like a bright enough PE comp control is not, in fact, true. The MFI (median) of the PE+ comp control is 1059, for the sample it is 1267. I used CD8 PE for both samples, but the comp control was about a week old (fixed mouse SC). We have been doing this pretty regularly the past year, but apparently a small loss of fluorescence over time, or differences in day to day staining, can make a big difference. I've included a graph with: Top = lymphocyte gate Middle = PE single stain (left), APC single stain (right) Bottom = CD8 PE v. IFNg APC left = uncompensated, right = compensated So my next question... do I even need to compensate PE v. APC? Obviously I can't do the biexponential transformation if I don't, but what are the rules about compensating channels with little or no spectral overlap? Thanks for everyone's comments. Diana Diana L. Martin Post-Doctoral Fellow Center for Tropical and Emerging Global Diseases University of Georgia Athens, GA 30602 (706)542-3396 This attachment - 'IC stain.jpg' - 357.04 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/29147e1f6be05cf6678981feeb4580106a11a184.jpgReceived on Fri Oct 7 13:38:00 2005
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