Culturing Synovial Fibroblasts and Stimulation

The human embryonal kidney cell line (HEK 293) (DMSZ, ACC 035) and HeLa (DMSZ, ACC57) cells were cultured in Dulbecco's modified Eagle medium (DMEM, Life Technologies, ThermoFisher Scientific, Waltham, USA) with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Life Technologies, ThermoFisher Scientific, Waltham, USA), 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mM sodium pyruvate (PAA the Cell Culture Company, Cölbe, GER). Synovial fibroblasts (SFs) of RA patients were cultured as described previously (16 (link)). This study was approved by the Institutional Ethical Committee (Solna, Stockholm, Sweden ethical number 2009/1262-31/3) and is in compliance with all ethical standards and patients' consent according to the Declaration of Helsinki. Cell culture was carried out in a humidified atmosphere of 5% CO₂ at 37°C. SFs were seeded at a density of 5 × 10⁴ cells/well in 24-well plates and cultured in DMEM-medium supplemented with EV-free FBS (Gibco, Life Technologies, ThermoFisher Scientific, Waltham, USA), 100 U/ml penicillin 100 μg/ml streptomycin and 1 mM sodium pyruvate for 24 h before they were stimulated with 10 ng/ml ml interleukin (IL)-1β (Sigma-Aldrich, Darmstadt, GER) or tumor necrosis factor α (TNF α) (Immuno Tools, Friesoythe, GER) for further 24 h.