Mice used were Foxp3-GFP knock-in B6/129 F2's of 5-8 weeks of age. Single cell suspensions of cells from spleens and lymph nodes of 25 male mice were generated. Cell suspensions were enriched for CD4+ T cells by magnetic antibody mediated depletion using a cocktail of biotinylated antibodies specific for CD8, B220, CD11b, NK1.1, and DX5 followed by anti-biotin MACS beads. The remaining cells were labeled with CD4-PerCP, CD25-APC antibodies, and PE-conjugated streptavidin. CD4+PE- cells were sorted into four populations based on expression of GFP and CD25. Total RNA was isolated and biotinylated antisense cRNA was prepared using two cycles of in vitro amplification according to the Affymetrix Small Sample Labeling Protocol II. Biotinylated cRNA was fragmented and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Keywords = regulatory T cells Keywords = Foxp3
