Profiling Synoviocytes under Inflammatory Conditions

Fibroblast-like synoviocytes (FLS) were generated from cRA SFMCs (33 (link)). FLS were evaluated at passages 3–4. Cultures were stimulated with interferon gamma (IFNγ) 10 ng/ml or TNFα 10 ng/ml (both from Peprotech) and subsequently with rhPD-1 (1 μg/ml) or left untreated as a control (34 (link)). Flow cytometry staining was performed after 48 h of stimulation, using anti-human antibodies, staining for CD90 FITC (BD, JJ, USA), PD-L1 PeCy7 (BD, JJ, USA), L/D nIR (Thermo Fisher, MA, USA), and RANKL PE (Biolegend). Gating was done on live cells, single cells, and CD90⁺ cells (Supplementary Figure S1). The flow cytometry was performed on the LSR Fortessa (BD), and data analysis in FlowJo. MCP-1 was measured by ELISA in the supernatant (Thermo Fisher, MA, USA).