Generation of M1 and M2 Macrophages from PBMCs

M2 and M1 macrophages were generated by the generally established methods [38 (link),39 (link)]. Human peripheral blood mononuclear cells (PBMCs) were collected from the peripheral blood of healthy volunteers using a BD Vacutainer^® CPT™ (BD Bioscience), and monocytes were isolated using a MidiMACS™ Separator (130-042-302, Miltenybiotec, Bergisch Gladbach, Germany), LS column (130-042-401, Miltenybiotec) and CD14 MicroBeads human (130-050-201, Miltenybiotec). CD14⁺ monocytes were cultured in a 12-well plate at a density of 5 × 10⁵ per well at 37 °C in 5% CO₂ atmosphere in RPMI1640 (Sigma-Aldrich) supplemented with 20 ng/mL of M-CSF (216-MC, R&D systems, Minneapolis, MN, USA), penicillin/streptomycin and 10% heat-inactivated FBS for 6 days. Subsequently, we continued to culture the cells in the same medium for 3 days to generate M0 macrophages, while we added 20 ng/mL each of IL-4 (204-IL, R&D Systems), IL-10 (217-IL, R&D systems) and IL-13 (213-ILB, R&D systems) into the culture medium and continued to culture the cells for 3 days for the generation of M2 macrophages. To differentiate M1 macrophages, we utilized 20 ng/mL of GM-CSF (7954-GM, R&D Systems) and 20 ng/mL each of LPS (L2630, Sigma-Aldrich), IFN-γ (285-IF, R&D Systems) and IL-6 (206-IL, R&D Systems).