Dear all. Yet another question, this time a little less related to flow cytometry itself - but I was hoping maybe anyone could halb me anyway: I am planning to use anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation in the context of whole PBMCs rather than purified T cells. However, I am a bit confused about the variety of protocolls out there (pre-coating plates with anti-CD3 and anti-CD28 VS pre-coating plates with anti-CD3 alone and then adding cells and soluble anti-CD28 VS pre-coating/staining cells with anti-CD3 and then resuspending in medium with anti-CD28 either immediately or after an initial 3-day culture with anti-CD3 alone). Also, there are two clones, OKT3 and UCHT1 - and according to some 1980s papers, all human donors respond mitogenic to OKT3, but not to UCHT1. I tested three donors stimulated with pre-coated UCHT1 and there was 1 strong, 1 weak and 1 non-responder. Given that I usually work with buffy coats from the bloodbank (which give me a hard time if I freeze them and use them after thawing (clumping, cell loss), so I tend to always use them fresh), I do not have the time to test each donor for being a UCHT responder. At the same time however, many companys such as BD don't even sell OKT3 but only UCHT1 and recommend the latter for T cell stimulation. Is there a reason for that? Hope some of you can share their experiences with me! Thanks, Anja _______________________________________________ Anja Scholzen PhD Student Burnet Institute at Austin Studley Road, Heidelberg 3084 VIC, Australia Tel.:0061 3 9287 0673 Fax: 0061 3 9287 0600Received on Mon Sep 11 14:58:00 2006
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