Generation and Maintenance of THP-1 and RAW 264.7 Macrophages

The human monocytic leukemia cell line THP-1 from the resource center of Peking Union Medical College Hospital (Beijing, China) and HEK 293T cells from Shanghai stem cell bank (Shanghai, China) were maintained in RPMI 1640 medium (Gibco) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Solarbio), 10 mM HEPES (Biosharp), and 10% fetal bovine serum (Gibco) at 37°C with 5% CO₂ in a humidified atmosphere. Prior to experiments, human monocytes (THP-1) derived macrophages (MΦ-THP-1) were generated by phorbol 12-myristate 13-acetate (PMA, 20 ng/mL) treatment for 48 h followed by resting cells for 12 h according to the methods in references (38 (link)–40 (link)). The mouse macrophage cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 10 mM HEPES, and 10% fetal bovine serum at 37°C with 5% CO₂ in a humidified atmosphere. All cell lines were cultured within 10 passages prior to use. gC1qR knock-down cell line (THP-1-sh-gC1qR) and control cell line (THP-1-sh-luciferase) were prepared as previously described (36 (link)). gC1qR-knockout cell line (THP-1-KO-gC1qR) were generated as described in Materials and Methods below.