By Annie Potter
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Extra resources for A Dozen Fabulous Pillows
Wash beads three times with Phosphate Buffer, centrifuging at 200 × g to remove buffer between washes. Count beads with a hemacytometer and resuspend beads at 5 × 107/ml in sterile Phosphate Buffer with 2 mM BSA. Optimal bead concentration is typically between 3:1 and 10:1 (T cell:bead ratio); however, this must be determined empirically by titrating beads into a proliferation assay prior to suppression assays. Desired Tconv cell proliferation is 40,000–80,000 cpm following 8 h [3H]-thymidine culture for the final 72 h of assay.
M. Blackman, J. Bill, J. Kappler, P. Marrack, D. P. Gold, and W. Born. (1989) Two better cell lines for making hybridomas expressing specific T cell receptors. J. Immunol. 143:1822–1825. 2. , M. Yamamoto, M. Takami, Y. Seki, M. Takezaki, A. L. Mellor, and M. Iwashima. CD4+CD25+ regulatory T cells resist a novel form of CD28- and Fas-dependent p53 induced T cell apoptosis J. Immunol. 184:94–104. 3. , J. Kern, N. Singh, M. Iwashima, P. Kraj, and L. Ignatowicz. (2007) Nonself-antigens are the cognate specificities of Foxp3(+) regulatory T cells.
Analysis of the CD4+ T cells revealed that the CD25+ regulatory T cell subset was significantly more activated in chronically infected mice than in naïve mice, the same subset of cells that Shimon Sakaguchi had shown to be involved in suppressing anti-self reactivity to prevent autoimmune diseases (7). These studies led to the development of in vivo and in vitro analysis techniques to further study the suppressive activity of virus-induced Tregs (4, 8, 9). 2. 1. Complete medium: Iscove’s modified Dulbecco’s medium (IMDM) (Lonza) with 25 mM Hepes, 10% heat-inactivated (56°C for 30 min) FBS, 100 U/ml penicillin and streptomycin, 2 mM l-glutamine.