Assuming you're talking about a three-color analyzer . . . looking at PI in the "FL3" (>650nm) channel will work OKAY. Keep in mind that you will see PI in "FL2" (~575BP) as well, but it's not a problem because you're excluding the cells that are PI positive, so they don't factor in. There can be an increase in noise to the PE channel, though, so it's probably good to compensate for it with single color controls. Anyway . . . yes, your approach is sound. I've even done the same looking at the PI signal in the PE channel, and eliminating the very brightest cells as the "dead" (PI+) cells. This can work on a machine with only two fluorescence PMTs. Alternatively, one can use 7-AAD as an exclusiond dye, and not worry so much about crossover, since the 7-AAD emits well into your "FL3" parameter's band range. MAK. Mark A. KuKuruga University of Michigan Flow Cytometry kukuruga@medmail.med.umich.edu
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