Differentiation and Characterization of Murine Macrophages

Bone marrow-derived macrophages from 9 to 12 weeks old ApoE^–/– and ApoE^–/–/Pad4^–/– mice were prepared by flushing femurs and tibias followed by cell culture in RPMI medium supplemented with 20% FCS and 20 ng/mL murine M-CSF (Peprotech) for 7 days (26 (link)). To induce a M1- or M2a-like phenotype, macrophages (M0) were further incubated in the presence of 20 ng/mL IFN-γ (Peprotech) and 100 ng/mL LPS (Sigma Aldrich) or in medium supplemented with 20 ng/mL IL-4 (Peprotech), respectively. Macrophages’ phenotype was characterized by Real-time PCR and flow cytometry (FACS Canto II, BD). For flow cytometry, M0, M1-like and M2a-like macrophages were stained for CD11b, CD86 (M1 marker) and CD206 (M2 marker). The following antibodies were used: APC-Cy7-conjugated anti-CD11b, APC-conjugated anti-CD86 and Brilliant Violet 421-conjugated anti-CD206 (BioLegend).