The experience of some people here (following a suggestion on this mailing list early) is that dyes with higher affinity than PI (7-AAD, TOTO-3, etc) can often maintain a distinction for considerably longer than two hours. Proper titration of some of these dyes may even allow a lyse/no-wash type of assay, low concentrations allow most of the dye to end up in the dead cells before fixation and the dilution with fixative keeps the remaining dye from effectively staining the cells that were alive. Tom Frey > Many moons ago I remember reading on the list a protocol > for discriminating viable cells from dead cells that > would still be usable after fixation of the sample. If you analyze within 2 hours of fixation with 0.5% PFA, then you can use PI: do standard PI staining, wash, then fix. If you wait more than 2 hours, the signal really degrades. The other alternative (which we have used for combining with intracellular staining) is to use EMA (ethdium monoazide). Stain cells with 5 ug/ml in the dark (20 min), wash twice, then expose to a UV source (like a UV light, even I think, a fluorescent light bulb). Wash. EMA is UV-crosslinked to become covalently bound to the cells and lights up cells that were dead before fixation and permeabilization. The EMA was published by a couple of people; see the Molecular Probes catalog for specific references. mr
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