Yes, we do this routinely using Ethidium Monoazide (EMA). EMA works like PI, with the bonus that when you expose the cells to light, the EMA covalently links to the DNA. Thus, the protocol is to incubate cells with EMA much like PI (in the dark) during the time when they are still alive, perhaps during staining, then wash them, then expose them to light for 10 minutes or so. Then you can fix & perm and do whatever you want to them. The only downside is that you must devote a whole channel to the EMA measurement (usually, FL3). EMA is not as bright as PI, so you can't overlay an immunofluorescence stain on top of the live/dead measurement as you can with PI. Although the EMA protocol was first published about 10 years ago, you can access our more recent protocol, where we optimized it for use with infectious PBMC & intracellular staining after stimulation: Mitra et al (1999). Differential representations of memory T cell subsets are characteristic of polarized immunity in Leprosy and atopic diseases. Intl. Immunol. 11:1801-1810. mr (PS, this same paper gives the protocol for doing CD4 immunophenotyping on stimulated cells where CD4 is internalized--i.e., prestain!) At 11:57 AM -0500 3/15/00, Zucker.Robert@epamail.epa.gov wrote: >At a recent flow cytometry workshop in the Research Triangle Park area the >following question was asked: Is it possible to measure viability in a cell >population and then fix the cells prior to analysis by flow >cytometry. Can the >dye stay in the dead cells without leaking into the live cells? I >remember that >Dr. Allan Pollock worked on these techniques in the 1980's . Does >any one have >any knowledge or information on this type of protocol. The research >groupe wants >to apply this technique on AIDS blood. Thanks >Bob > >Robert M. Zucker, Ph.D >U.S. Environmental Protection Agency >National Health and Environmental Effects Research Laboratory >2525 NC Highway 54, MD 72 >Research Triangle Park, North Carolina, 27709 >tel: 919-541-1585 fax 919-541-4017 >e-mail: zucker.robert@epa.gov
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