2.13. RAW 264.7 Macrophage Culturing and Treatment

RAW 264.7 macrophages were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco). The cells were incubated in a humidified atmosphere at 37°C with 5% CO₂. The cells were divided into three groups as follows: cells incubated with culture medium containing 0.1% DMSO (control group), cells stimulated with LPS (30 ng/mL; Sigma-Aldrich) (LPS group), and cells stimulated with LPS (30 ng/mL; Sigma-Aldrich) plus IL-10 (100 ng/mL; R&D Systems) (LPS+IL-10 group). After 24 h, the membrane surface molecules were stained with 3 μL/test of PE-conjugated monoclonal anti-mouse CD86 (Invitrogen) or APC-conjugated monoclonal anti-mouse CD206 (Invitrogen) for 30 min at room temperature in the dark according to the manufacturer's instructions.