Monocyte-to-Macrophage Differentiation and Polarization

The differentiation of monocytes to macrophages and polarization towards M1 and M2 was performed as previously described (Werz et al., 2018 (link)). M1 were generated by incubating monocytes with 20 ng/ml GM-CSF (Peprotech, Hamburg, Germany) for 6 days in RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml), followed by 100 ng/ml LPS and 20 ng/ml INF-γ (Peprotech) treatment for another 48 h. M2 were obtained by incubating monocytes with 20 ng/ml M-CSF (Peprotech) for 6 days and subsequent treatment with 20 ng/ml IL-4 (Peprotech) for additional 48 h. Correct polarization and purity of macrophages were routinely checked by flow cytometry (FACS Canto Plus flow cytometer, BD Bioscience) as previously reported (Werner et al., 2019 (link)) using the following antibodies: FITC anti-human CD14 (2 µg/test, clone M5E2, BD Bioscience), PE anti-human CD54 (1 µg/test, clone HA58, BD Bioscience), APC-H7 anti-human CD80 (0.25 µg/test, clone L307.4, BD Bioscience), PE-Cy7 anti-human CD163 (2 µg/test, clone RM3/1, Biolegend, San Diego, CA), and PerCP-eFluor710 anti-human CD206 (0.06 µg/test, clone 19.2, Biosciences, San Diego, CA).