Foxp3 expression in bone marrow cells and splenocytes of wild-type, Foxp3-GFP and Foxp3Cre Rosa-YFP mice in homeostatic and activating conditions. (A) Flow cytometric analysis was performed on bone marrow cells and splenocytes from wild-type, Foxp3^GFP and Foxp3^Cre RosaYFP mice (all on a C57Bl/6 background). Cells were stained with anti–CD11b-APC, anti–F4/80-PE or anti–CD4-PE. Percentages of single or double positive cells in the respective quadrants of the dot plots are indicated. Histograms show Foxp3 intensity in gated F4/80⁺CD11b⁺ cells. (B) Foxp3 mRNA levels were determined in macrophages and T cells by qRT-PCR. CD11b⁺ cell-enriched splenocytes were obtained by incubation with magnetic-activated cell sorting (MACS) CD11b beads and further purified after incubation with FITC-conjugated anti-CD11b antibodies and PE-conjugated anti-F4/80 antibodies and subsequent sorting by flow cytometry on a FACSVantage apparatus. CD4⁺ cells were obtained from lymph nodes and purified with the Mouse T cell CD4 Subset Column Kit. From this population, CD4⁺CD25⁺ Treg cells were purified by FACSVantage. The purity of the populations varied from 96% to 99%. Bone marrow cells were stimulated with 20 ng/mL M-CSF for 3 days. (C) Flow cytometric analysis was performed (as described in panel A) on bone marrow and splenocytes from wild-type and Foxp3^GFP mice, 5 weeks post immunization with collagen type II in complete Freund's adjuvant containing heat-killed Mycobacterium tuberculosis. (D) Bone marrow cells of naive mice were stimulated for 3 days with M-CSF (20 ng/mL) or M-CSF and IL-4 (10 ng/mL). Bone marrow cells of immunized mice were stimulated for 3 days with M-CSF and TGF-β (1 ng/mL). Cells were stained as described in panel A. Percentages of single or double positive cells in the respective quadrants of the dot plots are indicated. Histograms show Foxp3 intensity in gated F4/80⁺CD11b⁺ cells stimulated with M-CSF and TGF-β. In this condition, Foxp3^Cre Rosa-YFP mice were not included. In each of the panels, the data were obtained from a pool of 2 to 3 mice. ND indicates not detected.