Isolation and Characterization of Synovial Fluid Cells

Synovial fluid (SF) from inflamed knee joints was collected during active arthritis from 27 patients after obtaining informed consent. Patients’ characteristics are described in Table 1 and Supplementary Table S1. SF was centrifuged at 650× g for 20 min to pellet the cells. The SF was centrifuged at 3000× g for 30 min at RT to pellet all remaining cells and debris; the cell-free SF was collected and stored at −80 °C until further use. Meanwhile, the SF-derived cells collected after the first 650 g step were resuspended in IMDM (Gibco; Thermo Fischer Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS; Hyclone; Thermo Fischer Scientific Inc., Waltham, Mass) and gentamycin (86 µg/mL; Duchefa Biochemie B.V., Haarlem, The Netherlands) and passed through a 70 µm single-cell filter. Then, cells were resuspended in IMDM with 10% HI FBS at a concentration of 2 × 10⁶ cells/mL for flow cytometry analysis and culture experiments.