Flt 3

BMDCs were generated as previously described (25 (link), 30 (link), 31 (link), 33 (link), 34 (link)). Briefly, femurs and tibiae were collected from freshly euthanized B6 mice, BM harvested, and single cell seeded at 2 × 10⁵/ml. RPMI 1640 (Lonza) was supplemented with 10% FBS and 1% penicillin/streptomycin, plus 20 ng/ml mouse GM-CSF (all from BioLegend). For generation of CD103⁺ BMDCs, Flt-3 (100 ng/ml, eBioscience) was used instead of GM-CSF. On day 4, fresh GM-CSF (20 ng/ml) was added to both culture conditions.

Single cell suspensions of thymus, spleen, and bone marrow (BM) were harvested and counted at various time points. Cells from the spleen, and BM were subjected to ACK lysis to remove red blood cells. All flow cytometry specimens were incubated with anti-Fcγ III/II receptor (2.4G2) blocking antibody prior to staining. Samples were labeled using combinations of the following antibodies: CD4, CD8, CD3, CD44, CD25, B220, AA4.1, CD45.2, CD45.1, CD45, Sca-1, c-kit (APC (eBioscience) or PE), and IL7Rα (eBioscience), FLT3 (eBioscience), CCR9 (R&D systems), CD11c, CD31, Gr-1, Ki-67, Bcl-2 (Biolegend), CXCR4 (BD or Biolegend), CD150 (eBioscience), CD47 (Biolegend). For DN and ETP/LSK cells subset determination, mature cells were labeled with biotinylated lineage markers: TER, CD8α, H57, Gr1, Mac1, NK1.1, IgM, CD19, B220, CD3, and CD11c. Biotinylated antibodies were developed with streptavidan pacific blue (Invitrogen). All antibodies were purchased from BD unless indicated otherwise. Isotype controls were utilized for all rare populations, including florescence minus one controls. Four, five, and six color panels were acquired on a LSR II flow cytometer (BD Biosciences). All data was analyzed using FlowJo software (Treestar Software).