Hi Everyone: I have a person interested in looking at infiltrating leukocytes in the liver during inflammatory responses. I know there are many methods out there dealing with collagenase treatment, Percoll gradients, Ficoll gradients, etc. I know there is the potential to lose surface markers if you use the wrong or too much collagenase and that certain cells pellet (i.e.granulocytes) on gradients. I have tried various protocols in the past, but I was never quite sure if I was analyzing all the populations. Does anyone have a method that they use routinely use that you know works well, especially for studying the infiltrating leukocyte populations. Many Thanks, denise -----Original Message----- From: PAUL HALLBERG [mailto:Paul.Hallberg@mail.tju.edu] Sent: Thursday, September 12, 2002 2:27 PM To: cyto-inbox Subject: CFSE labeling for in-vivo vs. in-vitro experiments? I would appreciate any helpful information regarding CFSE labeling for in-vivo vs. in-vitro experiments. Below my client describes his results: In the in vivo experiments, I inject (intravenously) CFSE-labeled CD4+ T cells from C57B/6 mouse into bm12 mouse (allogenic setting). When I pull out the splenocytes and lymph nodes from the bm12 mouse and run flow analysis, the CFSE proliferation was not observed 48 h post-stimulation, but from 72 h onwards, CFSE proliferation was consistant (7 rounds of proliferation; undivided cells about 30%; precursor frequency about 14%). When I tried to repeat the same experiment in vitro, I see a prominant bunch of cells that are low on CFSE scale (corresponding to 4-5 rounds of division) in addition to the undivided (CFSE high) peak within 48 h. I do not see any ordered CFSE proliferation in vitro further on (after 48 h). My CFSE + mimosin and CFSE + ConA controls are very fine. We wonder whether synchronious stimulation in vitro is the reason for this observation, or whether I am missing something somewhere. Thanks in advance! Paul L. Hallberg Flow Cytometry Manager KCC CORE Flow Cytometry Facility Thomas Jefferson University 215-503-4556 215-923-0249(fax) Paul.Hallberg@mail.tju.edu *************************************************************** This message and any attachments is solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use, or distribution of the information included in this message is prohibited -- please immediately and permanently delete this message.
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