Since it sounds like you are interested in Th17 helper cells, I would definitely re-stain for CD4. Even if your purity is great from the column, how can you be sure some of those CD4+ cells aren't down regulating CD4 in that 5 day period? Some of your helper cells may not have bound antigen long enough and be losing their phenotype. And if the purity isn't ideal from column, you will want to re-stain anyways. Normally 100,000 cells should be statistically valid for cytokine data, but I would increase the volume and run it slow.... Eric O'Connor Head of Flow Cytometry MRC Clinical Sciences Centre Imperial College Faculty of Medicine Hammersmith Hospital Campus DuCane Road, London W12 0NN Tel: +44(0) 208 383 8330 Mobile: +44(0) 781 575 7730 ________________________________ From: Suat Dervish [mailto:sdervish@med.usyd.edu.au] Sent: 27 June 2008 05:11 To: cyto-inbox Subject: CD4+ T helper cell differentiation Hi flowers, I am doing some T helper differentiation experiments and am just wondering about some specifics that people are doing. If people magnetically select CD4+ T cells and then only culture these cells, for like 5 day later staining, do people restain for CD4 and then look at e.g IL17, IFN-Y production? Or do you only stain for these cytokines>?? And just choose everything that falls in the lymphocyte gate. What numbers of t cells are seeded to get good %'s when running flow. Example seeded 100000 cells / well. 200uL volume, is this usually enough? Because I am having trouble getting many events after all the staining, intracellular staining, etc etc. Any other ideas/useful heads upss would be greatly appreciated. Thanks --------------------------------------------------- Suat Dervish PhD Student The Sutton Arthritis Research Laboratories Royal North Shore Hospital NSW, Australia Phone:9926 6251 Email: sdervish@med.usyd.edu.au ---------------------------------------------------Received on Mon Jun 30 15:18:00 2008
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