Figure 4.

BCAP^−/− HSPCs produce increased numbers of myeloid cells in vitro. (A) Methylcellulose cultures were performed by sorting 250 LSK, CMP, or GMP cells from 3 individual WT and BCAP^−/− mice into methylcellulose containing SCF, IL-3, and IL-6. After 5 days, differential macrophage, granulocyte, and mixed macrophage/granulocyte CFUs were quantified. Data are pooled from 2 independent experiments with n = 6 mice per group. (B) At indicated days, cells were harvested from LSK, CMP, and GMP methylcellulose cultures and quantified by flow cytometry. (C) Absolute numbers of mature macrophages (left) and mature neutrophils (right) from WT and BCAP^−/− GMP methylcellulose cultures containing SCF, IL3, and IL-6 at various days after culture. (D) Absolute numbers of mature macrophages (left) and mature neutrophils (right) from WT and BCAP^−/− GMP methylcellulose cultures containing GM-CSF at various days after culture. (E) A total of 5000 LSK, 250 CMP, and 250 GMP cells were sorted from 3 individual WT or BCAP^−/− mice into methylcellulose containing SCF, IL-3, and IL-6. At indicated days, progenitor-derived cells were harvested from methylcellulose and cultured in media containing 10 μg/mL BrdU. Cells were incubated at 37°C in BrdU-containing media for 1 hour for LSK, and 4 hours for CMP and GMP. Cells were then harvested, fixed, stained, and examined for BrdU incorporation by flow cytometry. Frequency of BrdU⁺ cells from LSK, CMP, and GMP methylcellulose cultures at indicated days. In panels B through E, data are representative of 2 to 3 independent experiments; all graphs show mean ± SEM with n = 3 mice per group. *P < .05 and **P < .01, as determined by 2-tailed, unpaired Student t test.