> 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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