on 11/19/99 1:39 PM, Carla G Hill at Carla.Hill@bms.com wrote: > > I am asking for help in determining the conditions for > stimulating T cells with Staphylococcal enterotoxin B in Na > heparinized whole blood. I've tried 4and 6 hour incubation at > 37C using up to 40uL (10ug/mL) SEB, but no increase in CD69 > expression. > We routinely use SEB to study AICD in murine T cells, and I think the dynamics of activation are similar. We culture 1 x 10^6 cells (T cells and at least 1 class II positive APC population) and 1 ug SEB and 10U IL-2 per ml of RPMI (RPMI+10% FCS + PSN + 5 mM 2-ME) in 24 well plates at 1 ml per well (IL-2 is necessary for prolonged culture periods). We see CD69 expression as follows: 24 H: 28%+, 48 H: 47%+, 72 H: 57%+, and 96 H: 46% We analyze only the target T cell population (in mouse, mainly VB8+ T cells), and make sure we don't deplete the sample of APC's (SEB must bind class II on an APC to interact with the TCR). In humans, SEB will activate T cells bearing VB3, 12,14,15,17, and 20. Narrowing the population you analyze (using specific VB antibodies) will improve your sensitivity. Along the same lines, analysis of a non-target VB population can be used as a nice control. Hope this helps. -- Keith Bahjat Graduate Assistant University of Florida College of Medicine Gainesville, Florida Voice: (352) 392-4887 Fax: (352) 392-5393 e-mail: kbahjat@ufl.edu
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