Beads enriched CD 25 cells G 3 SSC

Beads enriched CD 25+ cells G 3 SSC G 2 STEP 3 Pre-enrichement and sorting FSC-H G 1 SSC-H FSC-H STEP 1 Doublet elimination G 1 x G 2 x G 3 (FSCLOWSSCLOW singlets) Beads enriched CD 25 - cells 30% 0. 2% CD 25 FSC-W SSC-W FSC-W STEP 4 – Purity of sorted cells SSC-A Leucocytes G 4 Sorted CD 250% 99% T cells CD 4+T cells G 5 G 6 CD 3 CD 25 STEP 5 – Foxp 3 Validation CD 25 Bright CD 4 CD 25 CD 45 SSC STEP 2 – CD 4+ T gating G 4 x G 5 x G 6 x G 7 (CD 45+CD 3+CD 4+Foxp 3+) Sorted CD 25 Bright 0. 1 98 0. 0 1. 9 0. 1 STEP 3 Foxp 3 Supplementary Fig. 1. Isolation and sorting Treg cells. Peripheral blood mononuclear cells and tissue mononuclear cells were stained for CD 4, CD 3, CD 25 and CD 45. The doublets were eliminated based on FSC and SSC and the size of live lymphocytes (Gates 1 -3, step 1). CD 4 + T cells were determined based on CD 45, CD 3, and CD 4 (Gates 4 -7, step 2). Then, CD 4+CD 25+ T cells were enriched with CD 25 beads (step 3) and sorted with high speeder with CD 25 bright cells and CD 25 - cells. The purity was verified by sampling staining (step 4). As we determined the expression of Foxp 3 in the small aliquots of the sorted cells from different donors, if Foxp 3 expression was less than 0. 5% in the sorted CD 4+CD 25 - T cells and higher than 95% in the sorted CD 4+CD 25 high T cells, these cells were utilized for functional assays.