We do that all the time, and the cells you buy from BD as control cells (Mick and Hick) are just that. Freeze in freezing medium (BD uses 90% serum and 10% DMSO; we use 20% serum, 10% DMSO, 70% any medium), thaw, wash, fix, et al. Alternatively--and I think this is better--fix your cells before freezing. At the Los Alamos course last June Carlton Stewart told our group that he fixes in methanol, and sticks in the freezer. I fix in 4% paraformaldehyde/DPBS, so I don't freeze in that after fixing. Beverly Barton "Reed, Doug S Dr USAMRIID" wrote: > I was just curious as to whether anyone has tried doing intracellular > cytokine staining on cells that had been frozen and then thawed. > Can this be done? What problems do we need to be on the lookout for? > > We are interested in doing some ICS as part of a pathogenesis study where we > will be taking samples from primates every day for six days. What we would > like to do is look at gamma interferon/TNFalpha production by lymphocytes > during the course of disease using whole blood similarly to what has been > published by Picker & others. My thought was to remove the red blood cells > and freeze the PBLs in freezing medium at -70 C. If we could freeze the > cells and do the assay at a later time, that would greatly ease the workload > during the study since other assays (looking for apoptotic cells, for > example) must be done "fresh" and constraints on bench space and time exist > as well. > > Your input would be greatly appreciated! > > Sincerely, > Doug Reed > > Douglas S. Reed, Ph.D. > Microbiologist > Department of Aerobiology and Product Evaluation > Division of Toxinology and Aerobiology > U.S. Army Medical Research Institute of Infectious Disease > 1425 Porter St. Ft. Detrick > Frederick, MD 21702-5011 > 301-619-6728 > 301-619-2541 fax > Doug.Reed@det.amedd.army.mil
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