Isolation and Purification of Thymocyte and Peripheral T Cell Subsets

Subsets of thymocytes were isolated by cell sorting as previously described [54 (link)], after cell surface staining using CD4 (GK1.5), CD8 (53–6.7), CD3ε (145-2C11), and CD24 (M1/69) (all from Biolegend). DP cells were CD4+ CD8 int/hi; CD4 SP cells were CD4CD3 hi, CD24 int/lo. Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53–6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4 + CD62LhiCD25-CD44lo were obtained by sorting (BD FACS Aria). Three cell isolations from independent mice were prepared for each of the three thymocyte subsets.