Isolation and Purification of Synovial Macrophages

Isolation of synovium was performed according to published methods (18 (link)–20 (link)). Briefly, immediately after euthanasia, the synovial tissues around the knee joints were collected and pooled from 3 animals per group. The synovium was minced and digested in a 1 mg/ml collagenase type I (Sigma-Aldrich) for 1 h at 37°C and rinsed through a 70-μm filter (BD Biosciences). The isolated synoviocytes were suspended in phosphate-buffered saline (PBS) containing 20 μg/ml of antibody cocktail. Brilliant Violet 510- and phycoerythrin-conjugated antibodies against mouse CD45.2 and F4/80 were obtained from Biolegend (Chaoyang, Beijing, China). For isotype control, Brilliant Violet 510- or phycoerythrin-conjugated non-specific mouse or rat IgG2a were substituted for the primary antibody, respectively. After incubating with antibody cocktails for 30 min at 4°C, the cells were washed with PBS and resuspended in PBS and macrophages were sorted using FACS and RNA was isolated from these sored cells.