Figure 35.

Gating strategy to sort CD4⁺CD25^hiCD127^lo Tregs from human peripheral blood. Lymphocytes were gated according to their size and granularity (A), doublets excluded (B) and live cells were gated (C). (D) CD4⁺ T cells from an enriched population of cells were gated during the cell sort. CD4 staining of total lymphocytes is shown for reference. (E) CD25^hiCD127^lo Tregs were gated from the CD4⁺ gate. In this representative plot, cells were enriched using the STEMCELL Technologies CD25-enrichment kit and subsequently stained with the CD25 (clone 2A3) PE Ab. CD25 and CD127 staining in CD25-depleted cells and non-enriched total lymphocytes are shown for reference. Data for panels A-E were collected on an LSR Fortessa X20. (F) CD25-enriched cells (Miltenyi kit) were stained with CD25 (clone 4E3) PE and one of four different CD127 Abs. Data were collected on a Beckman Coulter Moflo Astrios cell sorter (see Table 35).