Flow cytometry. The algorithm of regulatory T cells gating using PBMCs2 of the 74-year-old healthy donor. (a) Lymphocytes among PBMCs. (b–c) Establishing FOXP3+ gate using CD3- cells known not to express FOXP3: CD3- cells among lymphocytes (b); establishing FOXP3 (exon 1)+ gate using CD3- cells (c). (d–e) Detection of Treg: CD4+ T cells among lymphocytes (d); CD25+ FOXP3 (exon 1)+ – Treg among CD4+ T cells (e). (f–g) Analysis of FOXP3 isoform expression: all FOXP3+ cells among CD4+ T cells (f); calculation of FI (FOXP3 exon 2/total) parameter in all FOXP3+ cells = [(FOXP3 exon 2 MFI)/(FOXP3 exon 1 MFI)] (g). The solid arrows show the gating algorithm. The dashed bidirectional arrows show the alignment of the established gates for CD3- and CD3+CD4+ cells. 1a.u.: arbitrary units; 2PBMCs: peripheral blood mononuclear cells.