Agrin deficiency impairs monocytic cell maturation. (A) Representative flow cytometric analysis of the myeloid hematopoietic progenitors in control and agrin-deficient mice and relative frequencies among total BM cells (ctrl, n = 20; Musk-L;Agrn^−/−, n = 5). FACS analysis is referred to the Lin⁻/IL7R⁻/cKit⁺/Sca1⁻ gate, in which CMPs are defined as CD34^+/low/CD16/32^int, whereas GMPs as CD34⁺/CD16/32⁺. Equal numbers of sorted CMP(B) or GMP BM progenitors (C) and (E) or CD31⁺/Lin⁻ BM monoblasts (D) from P5 control and Musk-L;Agrn^−/− mice were cultured for 7 (B-C,E) or 5 days (D) with 50 ng/mL recombinant M-CSF (B-C,D) or with 20 ng/mL recombinant G-CSF (E) and total cell numbers relative to control were calculated. Data are from 2 (B-C,E) or 3 (D) independent experiments. Gate strategy was performed as shown in representative plots; R1: c-Kit⁺/CD31^high/Ly6C⁻/Ly6G⁻; R2: c-Kit⁺/CD31⁺/Ly6C⁺/Ly6G⁻; R3: c-Kit^low/CD31⁻/Ly6C^high/Ly6G⁻;R4: c-Kit^low/CD31⁻/Ly6C^int/Ly6G⁺. (F) Equal numbers of sorted Ly6C^high BM cells from P5 control and Musk-L;Agrn^−/− mice were cultured for 3 days with 50 ng/mL recombinant M-CSF and total monocytic cell numbers relative to control were calculated. Shown are a representative flow cytometric analysis of Ly6C^highF4/80⁻ (monocytes), Ly-6C^highF4/80⁺ (monocytes in the process of differentiating into macrophages), and Ly6C^loF4/80⁺ (mature macrophages) and the statistical analysis of the frequency of the 3 populations, (ctrl, n = 14; Musk-L;Agrn^−/−, n = 7). (G) Annexin V analysis on P5 BM monocytes and splenic macrophages (ctrl, n = 10; Musk-L;Agrn^−/−, n = 7;). In all panels, error bars represent SEM and *P ≤ .05, **P ≤ .01, ***P ≤ .0001.