Bone‐marrow‐derived macrophage isolation, culture and treatment

Briefly, primary bone‐marrow‐derived macrophages (BMDM) were obtained from the bone marrow of 6‐ to 8‐week‐old healthy C57BL/6 mice. After CO₂ anesthesia, mice were killed by cervical dislocation. After applying 75% alcohol to the skin, sterile tibia and fibula were obtained to isolate bone marrow, which was washed in complete RPMI‐1640 medium with a 1‐ml syringe three times and centrifuged for 5 min at 350 g. Cell pellets were suspended in complete medium, filtered through a 40‐μm cell strainer, centrifuged again with red blood cell lysis buffer (ACK lysis buffer), which lyzed red blood cells, and then resuspended in complete RPMI‐1640 medium containing recombinant mouse 40 ng/ml macrophage colony‐stimulating factor in tissue culture dishes at a density of 2 × 10⁶ viable cells per plate and the plates were placed in a 37° incubator with 5% CO₂. At day 3 the culture plate was gently shaken, then medium was replaced with fresh medium (to replenish cytokines, and remove granulocytes and lymphocytes), and medium was then changed every other day. On day 7, BMDMs were collected, washed twice and suspended in an appropriate amount of complete RPMI‐1640 medium for further use.