IL-6, IL-1β and TNF production measurement by ELISA

The synergistic effect of IL-17 (50 ng/ml) and TNF-α (500 pg/ml) on the production of IL-6 by synoviocytes was already evaluated in previous papers [11,12]. Here a dose-response curve was established, comparing the known effect of IL-17/TNF combination to the one of IL-17 in association with IL-1β (10 and 100 pg/ml). IL-1β was first used alone or in association with IL-17 to verify the synergistic effect.

IL-6 production was quantified in synoviocyte and co-culture supernatants of day 5 by standard ELISA techniques (R&D system, San Diego, CA, USA). IL-1β and TNF-α production was measured in PBMC and monocyte cultures or in co-cultures in the presence or not of Cd. Absorbance at 450 nm was measured using the VICTOR X4 plate reader and results were obtained by subtracting the background read at 540 nm and compared to the regression curve obtained by standards of R&D system kit following manufacturer instructions.