Hello, everyone: One of our graduate students is trying to develop an assay for enumerating mouse splenocytes which are activated in response to a particular antigen. He is using anti-IFN-gamma production to identify activated cells. His protocol requires approximately 1 week in cell culture, during which time most non-activated splenocytes die (a majority of the starting population of cells). This is followed by a short period of restimulation with the antigen and staining for IFN-gamma. Flow cytometry analysis is hampered by the large population of dead cells (not to mention that the dead cells are probably soaking up his monoclonal antibody during staining). He is reluctant to use a Ficoll-Hypaque gradient to clean up the dead cells for fear that this would result in non-specific activation of his cells. Do any of you have a method of cleaning up the dead cells prior to cell staining that would not result in activation of the viable cells? Thanks in advance, Rick Meister * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Richard K. Meister Email: meister.1@osu.edu * * The Ohio State University Voice: (614) 292-9716 * * Dept. of Veterinary Biosciences FAX: (614) 292-6473 * * Cytometry Instrumentation Lab * * 1925 Coffey Road * * Columbus, OH 43210 U.S.A. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
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