The incompletely differentiated monocytes that accumulate in IRF8-deficient mice have leukemic properties. (A) Incompletely differentiated monocytes (CD11b⁺Ly6G⁻CD115⁺Ly6C^int) in the bone marrow and spleens of wild-type and IRF8^−/− mice were detected by flow cytometry (see supplemental Figure 11 for full gating strategy). Bone marrow numbers are per leg (femur + tibia). Data are representative of 5 mice per group. Statistical significance was assessed by Student t test (**P < .01, ***P < .001). (B) CD11b⁺Ly6G⁻CD115⁺Ly6C^hi monocytes (wild type) and CD11b⁺Ly6G⁻CD115⁺Ly6C^int cells (IRF8^−/−) were isolated from bone marrow and their morphology was assessed using May-Grünwald Giemsa staining. (C-E) CD11b⁺Ly6G⁻CD115⁺Ly6C^int cells were isolated from the bone marrow of IRF8^−/− mice by FACS sorting and labeled with CFSE. One thousand cells per well were cultured in liquid media containing M-CSF for the indicated time points. (C-D) Their ability to differentiate was assessed by flow cytometry; cells presented in panel C are CD11b⁺. (E) CFSE dilution was measured by flow cytometry to assess proliferation of the CD11b⁺Ly6G⁻CD115⁺Ly6C^int cells. Three wells per time point were pooled for analysis. Data are representative of independent cultures of CD11b⁺Ly6G⁻CD115⁺Ly6C^int cells from 3 IRF8^−/− mice.