Measurement of gene expression by enzyme-linked immunosorbent assay
Synovial cells (2.5 × 105 cells per 60-mm dish per 2 mL of serum-free media) were treated with recombinant adiponectin (1 or 10 μg/mL) or IL-1β (0.1 or 1 ng/mL) (ProSpec, Rehovot, Israel). Conditioned media was collected 24 hours later. Briefly, FLS cultures were centrifuged and the supernatants were collected and analyzed for IL-6, IL-8, PGE2, VEGF, MMP-1, and MMP-13 with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN, USA). Three independent experiments were performed in quadruplicate. Each experiment was performed using synovial cells from different patients. For the assessment of MMP-1, MMP-13, VEGF, and adiponectin levels in joint fluid, the collected joint fluid from 30 patients with RA or OA was dispensed into 1-mL aliquots and treated with hyaluronadase at 50 μg/mL for 1 hour at room temperature. The joint fluid was diluted with diluent's buffer for the proper detection range with ELISA. The levels of proteins of interest in joint fluid were measured using a commercial ELISA kit from R&D Systems, Inc., as described above.