Additional file 4: Supplement-Figure 4. Old mice overall possess less FoxP3- Th cells, but more Helios+ FoxP3- Th cells. a-f) Spleens, thymi or peripheral LN (inguinal, brachial, axillary) cells from young (average age of 18 weeks) and old (average age of 101 week) mice were collected. a) Total CD45+ cell counts are summarized (n=8/ group). b, c) Thymocytes (n=8 mice/ age group) were stained intracellularly for FoxP3, CD4, CD8 and CD45. CD4+ or CD8+ single-positive cells among CD45+ thymocytes were analyzed as represented in the FACS plots and summarized in the diagrams (b). Frequencies of FoxP3+ or FoxP3-CD4+ cells among CD45+ thymocytes are summarized in (c). d, e) Splenocytes (d) and peripheral LN cells (e) were stained as described in (c). Data are summarized in the box plots (n=8/ group). f) Peripheral LN cells were intracellularly stained for CD45, CD4, FoxP3, RORγt, GATA3 (all n=8) and Helios (n=5). Frequencies of the indicated FoxP3-CD4+ populations among CD45+ cells are shown. Statistical testing was performed as described in the methods section.