General responses for the PE-Viability issue. . . 1) PI works well as a viability marker, as most know. With a low concentration of PI, it's easily separated from PE. 2) 7AAD also works well, as one other message indicated, particularily since the resultant fluorescence is >650. Both are excluded from live cells, and serve well to detect damaged or dead cells. On FDA . . . 1) while FDA is a good indicator of esterase activity, it's not necessarily the best "viability" indicator. The free fluorescein resulting from the metabolism of FDA can readily leak out of cells, thus indicating false "dead," and also can stain non-specifically, thus adding to background -- so the carboxy form is recommended. 2) I've seen a similar compound -- Molecular Probes' calceim AM viability dye -- actively "pumped" from cells expressing p-Glycoprotein 170, so "green-negative cells" are not necesarily "dead." Usually, when dyes such as FDA are employed as a viability marker, PI has been used (quite necessarily) in combination to confirm the results. MAK. Mark A. KuKuruga University of Michigan Flow Cytometry kukuruga@medmail.med.umich.edu
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