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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2011 Jan 9;17(3):309–318. doi: 10.1016/j.bbmt.2010.12.710

Fig. 1. Isolation of iTregs and nTregs.

Fig. 1

A, generation and purification of iTregs. CD4+CD25− cells were purified from spleen and lymph node of OT-II TCR Tg and foxp3/gfp KI mice. These purified T cells were stimulated with OVA peptide at 0.5 μM in the presence of irradiated TCD-splenocytes. TGFβ was added in the culture at 2 ng/ml for Treg generation. Four to six days after culture, cells were harvested and stained for CD4, CD25 and GFP expression. The phenotype of cultured cells is shown on gated live cells (2 left panels). CD4+ CD25+GFP+ and CD25+GFP− cells were separated by FACS sorting (2 right panels). B, purification of nTregs. CD4+ cells were isolated through negative selection from spleen and lymph node of B6 foxp3/gfp KI mice. These CD4+ enriched cells were stained for CD4 and CD25 expression. The phenotype of these cells is shown on gated live cells (2 left panels). CD4+ CD25+GFP+ and CD25-GFP− cells were separated by FACS sorting (2 right panels).