Isolation and Culture of Mouse Bone Marrow-Derived Macrophages

We used 3-week-old C57BL/6J mice for cell extraction and obtained approval from the Animal Ethics Committee of Qilu Hospital, Shandong University (DWLL-2021-136). After administering Pelltobarbitalum Natricum (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) to euthanize the mice, we carefully isolated the femur and flushed the bone marrow cavity using the basal medium. We used RPMI-1640 and DMEM/F12 (1:1) medium to culture BMDMs and BMMSCs respectively. 10% Fetal Bovine Serum (FBS), and 1% antibiotics were added to the basal medium. Bone marrow cells were cultured for 7 days to obtain BMDMs by adding 50 ng/mL M-CSF (#216-MC, R&D System, Minneapolis, MN, USA) to RPMI-1640 as previously described [18 (link)]. Subsequently, 20 ng/ml IL4 was added and incubated for 24 hours to induce differentiation of BMDMs to M2 macrophages. We examined M2 macrophage differentiation using flow cytometry. Place the cells in the incubator and set the temperature to 37°C. The medium was changed regularly and passaged after digestion using trypsin (#25200056, Gibco, Rockville, MD, USA). Cells within 5 generations were selected for the experiment.