Fitc cd64

The cultured synoviocytes were harvested by 0.25% trypsin for 5 min and adjusted to 1.0 × 10⁷ cells/mL in DMEM/10% FBS. A 100 μL FLS suspension was transferred into 1000 μL PBS/10% FBS and centrifuged at 900× g for 3 min. Cell pellets were resuspended in 100 μL PBS/10% FBS. Amounts of 5 µL of FITC-CD90, FITC-CD64, PE-CD55 and APC-CD11b (Biolegend, San Diego, CA, USA) were added, and after the vortex, cell suspensions were incubated at room temperature (RT) in the dark for 30 min. PBS/10% FBS was added at 1000 μL and centrifuged at 900× g for 3 min. Cell pellets were resuspended in 500 μL PBS/10% FBS and fixed in 500 μL PBS containing 4% paraformaldehyde at RT for 15 min in the dark. Cell suspensions were pelleted again and finally resuspended in 1000 μL PBS/10% FBS for flow cytometry: BD LSR II (San Jose, CA, USA). When the homogeneity of synoviocytes was >95% CD90+, ≥90% CD55+, <1% CD11b+ and <1% CD64+, the FLS were used for the following experiments (Figure S1).

Single-cell suspensions were prepared from the spleen and VAT and stained with PE-MHC-II (107613, Biolegend), APC-cy7-CD11c (117323, Biolegend), FITC-CD64 (139315, Biolegend), Percp-cy5.5-CD45.2 (109827, Biolegend), APC-CD45 (147707, Biolegend), FITC-CD4 (100405, Biolegend), APC-CD8 (126613, Biolegend), PE-cy7-CD62L (104417, Biolegend), Percp-cy5.5-CD44 (103031, Biolegend), APC-cy7-FVD, or Pacific blue-DAPI. Intracellular staining was performed using PE-Foxp3 (12-5773-82, eBioscience) and antibodies against cytokines, including BV421-CD8, PE-cy7-IFN-γ (505825, Biolegend), Percp-cy5.5-IL-17A (506919, Biolegend), and APC-IL-4 (504105, Biolegend). Cells from the spleen and VAT were treated with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (500 ng/mL) (Sigma-Aldrich) for 4.5 h. Then the cytokines in specific cell populations were analyzed. The cells were stained at 4 °C for 30 min or 1 h for surface and intracellular staining, respectively, followed by detection using the FACS Canto II (BD Biosciences) system and analysis of the data using FlowJo (Tree Star, version 10.0).