Macrophage Differentiation and Culture

The human monocytic leukemia cell line THP-1 (resource center of Peking Union Medical College Hospital, China) and HEK 293T cells (Shanghai stem cell bank) were cultured in RPMI 1640 (Gibco) containing penicillin-streptomycin (100mg/mL for each drug, Solarbio), 10mM HEPES (Solarbio), 0.05mM 2-mercaptoethanol, and 10% fetal bovine serum (Gibco) at 37°C with 5% CO₂ in a humidified atmosphere. Before each test, monocytes (THP-1) were differentiated into macrophages (MΦ-THP-1) after treated with phorbol 12-myristate 13-acetate (PMA, 15ng/mL, multiscience) for 48h. Mouse BMDMs were isolated and cultured using standard protocols [46 (link), 47 (link)]. Bone marrow cells from BALB/c mice were collected by flushing the femurs from six-to eight-week-old mice with phosphate buffered saline (PBS) and then cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (BI) and macrophage-colony-stimulating factor (M-CSF) (Peprotech) at 37°C with 5% CO₂ for 7 days. After 7 days of culturing, non-adherent cells were eliminated. Adherent cells, which were highly enriched in BMDM, were recovered for studies. All the animal procedures were vetted and approved by the Hebei Medical University Ethical Committee for Animal Experimentation.