Lin⁻Sca-1⁻c-Kit⁺integrin β7^hiCD16/32^hibone marrow cells constitute bipotent mast cell/basophil progenitors. Bone marrow cells from WT mice were analyzed by flow cytometry and bipotent BMCPs, MPs, and GMPs were sorted and cultured in myeloid promoting-conditions or in erythroid-promoting conditions. (A) The gating strategy of primary BMCPs, MPs, and GMPs and the experimental setup are shown. The frequency of BMCPs is indicated as percentage of Lin⁻Sca-1⁻c-Kit⁺ cells. (B) Sorted cells were cultured in myeloid-promoting conditions for 5 days and stained with May-Grünwald Giemsa. Photo width, 33 μm. Images were captured using the Axio Imager.Z2, Axiocam 506, and Zen software (Zeiss). (C) BMCPs, MPs, and GMPs were cultured in bulk with myeloid-promoting cytokines and analyzed by flow cytometry. Supplemental Figure 4B shows the gating strategy. Day 7 cells were analyzed without the CD49b antibody. (D) Colonies derived from single BMCPs, MPs, and GMPs cultured in myeloid-promoting conditions were analyzed with flow cytometry. Supplemental Figure 4E shows the gating strategy. (E-F) BMCPs and MEPs were sorted and cultured in erythroid-promoting conditions. The cultured cells were analyzed by flow cytometry. Bulk cultures (E) and single-cell cultures (F) are shown. Supplemental Figure 5B shows the gating strategy. Panels C and D show data pooled from 2 experiments per time point from 4 independent sorts. Panels E and F show data pooled from 2 independent experiments. The number of single-cell colonies in which the cell types were determined is indicated above each bar. Supplemental Figures 4F-H and 5C-D show the colony sizes. Bulk indicates 50 to 100 sorted cells. Bone marrow cells were pooled from 2 to 4 mice per experiment. Ba, basophil; Bl, blast; Eo, eosinophil; Ery, erythroid; MC, mast cell; N, neutrophil.