Isolation and Culture of Bone Marrow Neutrophils and Macrophages

Bone marrow neutrophils were separated according to references [35 (link)]. Briefly, C57BL/6 mice were sacrificed, and the tibia and femur of the mice were removed. The bone marrow was washed out and dispersed into single cell suspension. Three milliliters of Histopaque-1119 (SIGMA, 11191), 3 ml of Histopaque-1077 (SIGMA, 10771), and the cell suspension were sequentially added into a 15 ml centrifuge tube to be centrifuged (700 g, 30 min, brake off) to have stratification. The cells between the 1119 and 1077 fluid layers were collected as polymorphonuclear neutrophil. After being washed with HBSS twice, neutrophils were labelled with the CFDA SE Cell Proliferation and Tracer Detection Kit (Beyotime, C0051) and then transferred to DMEM/HIGH GLUCOSE (HyClone, SH30022.01) complete medium to be cultured for 24 hours to induce apoptosis [36 (link)–38 (link)]. Similar to the above method, the isolated bone marrow cells were cultured in RPMI complete medium. One day later, suspension cells were collected and cultured for at least 6 days with 100 ng/ml macrophage colony-stimulating factor (M-CSF) (PeproTech, 315-02) to obtain BMDM. All cells were cultured at 37°C under 5% CO2 and 10% FBS (Gibco, 10099-141), and 1% Penicillin-Streptomycin Solution (HyClone, SV30010) was added.