Yes, it is 90% FCS. It comes from the Pharmingen catalog Cytokine Analysis protocol under "5. Alternative Fixation and Permeabilization Protocol". It does fix before staining so surface abs have to be able to withstand the fixing process. --On Tue, Aug 31, 1999 10:30 PM -0400 Sharon Vogt <svogt@bellsouth.net> wrote: > >> 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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