Bob, Use ethidium monoazide (EMA) from Molecular Probes. Easy as can be, you add 10ul of a 5ug/ml solution to 10E6 cells, incubate in the dark 10-20 minutes, put them under a desk light (2 foot fluorescents work great, but any kind will do) for 10-20 minutes and then add the add your flow reagents. EMA binds to the DNA of dead cells and will not release once cross-linked with the light. It excites with 488 and emits in the FL3 channel. There is no need to wash the cells after the EMA until the tubes are washed after the primary antibody. Randy Fischer TherImmune Research Corporation 9700 Great Seneca Hwy Rockville, MD 20850 (240) 453-6256 RFischer@therimmune.com > ---------- > From: Zucker.Robert@EPAMAIL.EPA.GOV > Sent: Wednesday, March 15, 2000 8:57 AM > To: Cytometry Mailing List > Subject: viability on fixed cells > > > > > 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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