Cell-surface phenotypic, morphologic, and growth properties of 4-1BB^-/- mast cells. (A) Wt and 4-1BB^-/- BMMCs were generated from bone marrow cells in IL-3-containing culture medium. Cohorts of 3 to 5 mice in each group were used to generate BMMCs. Results shown in this figure are representative of at least 3 independent experiments. Surface expression of FcϵRI and c-Kit was analyzed by flow cytometry. (B) Toluidine blue-stained wt and 4-1BB^-/- mast cells. Images of the cells at a magnification of × 1000 were acquired with a Nikon Optiphot photomicroscope (Nikon, Tokyo, Japan) with a Plan Apo 100 ×/1.40 NA oil objective, using a DVC-1310 camera (DVC, West Austin, TX) and DVCC-View v2.2 software. (C) Growth curves of bone marrow cells derived from wt (□) and 4-1BB^-/- (▪) mice in IL-3-containing medium. Each growth curve was generated from cultures in 6 flasks, each containing bone marrow cells derived from 1 femur (left or right) of 3 wt or 3 mutant mice. Similar results were obtained in 3 independent culture series. ^*P < .05, ^**P < .01, ^***P < .005 (versus wt control, Student t test). (D) Wt (□) and 4-1BB^-/- (▪) mast cells that had been deprived of growth factors for 8 hours were incubated with the indicated concentrations of IL-3 and SCF for 12 hours. DNA synthesis was measured by [³ H]thymidine incorporation during the last 6 hours of culture. ^*P < .05 (versus wt, Student t test). (E) Wt (□) and 4-1BB^-/- (▪) mast cells were incubated without IL-3 or other growth factors for the indicated periods. Live cells were quantified by flow cytometry of annexin V- and propidium iodide-stained cells. Annexin V-negative and propidium iodide-negative cells are plotted as function of incubation time. For panels C-E. Values shown are mean ± SD.