Comparative quantification of GFP⁺ DP cells.

Flow cytometric isolation of GFP⁺ DP thymocytes from (A) Foxp3^GFP, BAC-Foxp3^Cre-GFP and (B) Foxp3^IRES-GFP mice. Representative dot plots (from left to right) show presort analysis of CD4/CD8 expression among total thymocytes and CD25/GFP expression among CD25-enriched populations of gated DP cells, as well as postsort analysis of CD25/GFP and CD4/CD8 expression after flow cytometric isolation according to sort gates for CD25-enriched CD25⁺GFP⁺ cells, as indicated. Lines with arrowheads illustrate the gating scheme. Numbers in dot plots indicate percentages of cells in the respective quadrant or gate. (C) Quantification of GFP⁺ thymocytes. Percentages (left) and numbers (right) of GFP⁺ DP cells (top) and GFP⁺ CD4SP cells (bottom) from indicated Foxp3 reporter strains, revealed after postsort analysis as depicted in (A,B). All mice were six weeks old. (D) Numbers of GFP⁺ DP thymocytes from eleven-week-old Foxp3^GFP mice on the C57BL/6 (left) and BALB/c (middle) genetic background, as compared to age-matched BAC-Foxp3^Cre-GFP mice (right). Dots and horizontal lines represent individual mice and mean values, respectively. * p < 0.05, *** p < 0.001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison post test).