> Stan, I have a user who routinely freezes his cells prior to staining for > surface markers and cytokines. However, he does not freeze the whole blood > as you asked. He processes the blood as he would for non frozen samples - > stimulates, lyses, fixes - then suspends them in 10% DMSO and 90% FBS and > freezes. When he's ready to run, he thaws and stains. Hi all, In the above scenario, the cells are fixed before freezing. While certain antibodies (to surface antigens) will bind after fixation, others won't - true? Is it really 90% FBS? I should start by saying I don't have _much_ experience with this, but have followed some protocols for freezing wbc from a ficoll prep with DMSO (8-9%) in PBS w/ some small percentage of FBS. I limit the number of cells per ml to 1-2x10e7, thinking that some limit is necessary, but arbitrarily picking that range. Assuming that all of that is ok, why can't you do the same thing with whole blood? It must not be as simple as the freezing or thawing process acting to lyse the rbc, because the question is raised from time to time. Is there too much protein or cellular material released? Since grans don't tolerate freezing very well, the percentages of wbc populations would be altered, but mononuclear cells would be alright? Maybe I'll try it, but I don't really have the need to since we don't routinely freeze cells in my lab - I'm just curious. If I'm completely missing some basic concept, please feel free to let me know. Thanks, sharon Sharon F. Vogt Dekalb Med. Center Atl., GA
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