Hi Michal: TCR are down-modulated on activated T cells, so it's unlikely you'd see double-positives (tetramer+/IFNg+) after 48 hours of stimulation. For an example of simultaneous tetramer and intracellular cytokine staining, see: Viral Immunology 14(1):59-69, Direct Functional Analysis of Epitope-Specific CD8+ T Cells in Peripheral Blood, Xiao-Song He, et al. 2001. Barry Barry Bredt Core Laboratory Director UCSF/SFGH General Clinical Research Center Phone 415-206-5210 Pager 415-719-0339 Fax 415-206-8200 barryb@itsa.ucsf.edu Mail - UCSF Box 1353, San Francisco, CA 94143-1353 Shipments - SFGH Bldg 100, Room 249, 1001 Potrero Av, San Francisco, CA 94110 >Dear experts in flow cytometry, > >I would like to perform INF g and TNF b intracellular staining of a >small (0.1%) tetramer + PBMC. >I have some doubts and I would be grateful if you could give me any >suggestion. > >My lymphocytes are frozen and I wonder if there are any particular >conditions for its stimulation with specific peptide. > >The other thing that bothers me is the precision and specificity of >facs analysis. My population of Tetramere + cells is already small >and its subpopulation of INF g + will be even more difficult to >distinguish from the background staining. I wonder if it would be >possible (and correct) to culture the lymphocytes after peptide >stimulation during 48h to increase the number of double positive >cells without changing considerably the proportion of tetramer+/ INF >g + cells? > >Thanks for any suggestions! > >Michal >-- >Michal Abel, MD, PhD >Laboratory of Clinical Immunology >INSERM U25 >Necker Hospital, 161 Rue de Sèvres. Paris >tel: 33 1 40 19 28 87 >fax: 33 1 44 49 53 74
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