SCF-dependent activation of cS5^F promotes the development of murine mast cells in vitro. (A) Bone marrow (BM) cells from cS5^F mice were cultured in presence of SCF (10 ng/mL) or Flt3L (10 ng/mL) and viable cells were enumerated daily using the trypan blue dye exclusion method. (B) cS5^F-BM cells were deprived of SCF for 3 hours and then restimulated for the indicated times. Cell extracts were prepared and analyzed by Western blot with anti–P-Y-Stat5 and anti–Stat5 antibodies. (C) Two-week-old cS5^F-BM cells grown in culture in presence of SCF were analyzed for expression of FcϵRI and c-Kit by flow cytometry. (D) cS5^F-BM–derived MCs (cS5F-BMMCs) were incubated with anti-IgE alone or/and IgE. Alternatively, cells were treated with DMSO or A23187 and stained with toluidine blue. (E) cS5^F-BMMCs and GFPv-BMMCs were treated or not with LY294002 (1 μM) for 3 days and the percentage of viable cells was determined daily with the trypan blue dye exclusion assay. Results shown are representatives of 3 independent experiments.