TNF promotes the differentiation of Ly6C⁺Gr1^-CD11c⁺ M1 macrophages.

<p>(A) Freshly isolated bone marrow cells (BMCs) from 3-month-old C57Bl6 mice were stained with anti-mouse APC-Ly6C, PEcy7-CD11b, PE-Gr1, FITC-CD11c and PEcy5-F4/80 antibodies and expression levels of these cell surface markers were analyzed by flow cytometry. (B) BMCs (2x10⁶) from the mice in (A) were cultured with M-CSF, M-CSF+TNF (20ng/ml) or M-CSF+RANKL (10ng/ml) in 60 mm-dishes for 3 days to recruit OCPs, which we called M-CSF-induced OCPs (M-OCPs), TNF-induced OCPs (T-OCPs), and RANKL-induced OCPs (R-OCPs), respectively. IFN-γ (1ng/ml) was also added to M-CSF-treated cells as a positive control for M1 macrophage recruitment. Cells attached to the dishes were collected and stained with the above antibodies to analyze expression of cell surface markers by flow cytometry: CD11b⁺F4/80⁺ cells in the total cultured OCPs (upper panel), Ly6C⁺Gr1^- and Ly6C^-Gr1^- cells in the CD11b⁺F4/80⁺ population (middle panel) and CD11c⁺ cells in the Ly6C⁺Gr1^- and Ly6C^-Gr1^- populations (lower panel). The experiment was repeated three times with similar results.