Isolation and Characterization of Mouse Peritoneal Macrophages

PM were isolated as described previously [41 (link)]. Briefly, about 5 mL of cold phosphate buffer saline (PBS) was injected into mouse abdominal cavities. After vigorously shaking the mice for 2 min, solution in abdominal cavity (the PBS containing PM) was carefully collected. PM was obtained by centrifugation at 1000 g for 5 min. For flow cytometry analysis, equal amounts of the PM cells (1 × 10⁶ in 100 μL PBS) were incubated with appropriate antibodies; PE anti-mouse F4/80 antigen (eBioscience, San Diego, CA, USA), FITC anti-mouse CD11c antigen (BD Bioscience, San Jose, CA, USA), and APC anti-mouse CD206 antigen (BD Bioscience). The flow cytometry data were collected using a FACScan and analyzed with Cell Quest software (BD Biosciences). Macrophages labeled with F4/80⁺CD11c⁺CD206⁻ were counted as pro-inflammatory M1-like macrophages and those labeled with F4/80⁺CD11_C⁻CD206⁺ were counted as anti-inflammatory M2-like macrophages.