I am scratching my head after spending the last hour with clients who want to use 7-AAD to measure apoptosis and also stain with FITC and PE to measure various monoclonals. When we run their cells with the 7-AAD stain only we get nice differentiation of live, apoptotic and dead cells. When I then run a sample stained with FITC only on the 7-AAD program the fluorescence mesured on the 7-AAd PMT (PMT 3, 625) goes way up. Of course it does, FITC needs to be compensated out. When I go to my color comp controls, nothing I do alters this problem. I got so frustrated I turned up the FL-1 and FL-3 color comp components all the way up and nothing changed. So what's up here? The same "phonomenon" occurred when I ran a PE only stained sample. The 7-AAD fluorescence changed considerably from a sample run with 7-AAD and a sample stained with PE only, with no response from the color comp controls. I know the color comp controls work because earlier in their visit we set up the FITC/PE samples and the color comp did it's job. Any suggestions? Does what I described make any sense, or am I rambling? It's very possible because I am at a loss right now. Any and all suggestions at how to handle this phenomenon would be appreciated. Thanks in advance, Joan Kalnitsky Joan Kalnitsky Flow Cytometry Laboratory VMRCVM (540) 231-4115 FAX 540-231-7367 "It is better to serve, than to receive." B. Borg
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