Dear Rick, Try using a metrizamide gradient, this is supposed not to activate cells. Our recipe: 18% metrizamide gradient. Prepared as follows: 1) Stock of 35.3% metrizamide w/v into distilled water. 2)Working soloution: 1.02ml stock + 0.94ml of PBS +0.04% Fetal calf serum. Prepare 1ml of 18% metrizamide for 1ml of cell suspension, at 1*107 cells /ml. 3)Underlay cells with metrizamide, spin at RT, 1500 rpm for 15mins, no brake. Karin Haverson On Tue, 14 Nov 2000 18:04:26 -0500 "Richard K. Meister" <meister.1@osu.edu> wrote: > 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. * > * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * > ---------------------- Dr.K Haverson, Division of Clinical Veterinary Science, University of Bristol, Langford, BS40 5DU, UK e-mail: Karin.Haverson@bristol.ac.uk Tel.: (44) 117 928 9289 FAX: (44) 117 928 9505
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