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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
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Phone: (765)-494-0757;
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