Quantification of Inflammatory Cytokines

Culture supernatants from cells treated with mAb plus adiponectin were analyzed with IL-6 and IL-8 ELISA kits (BD Bioscience Korea, Seoul, Korea) in accordance with the protocol of the manufacturer. To screen hybridoma clones for mAbs against adiponectin, 96-well plates were coated with adiponectin (200 ng/well), hybridoma culture supernatants were added after blocking, wells were incubated with a 1:1,000 dilution of horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich Korea), and the ELISA procedure was completed. Sera from CIA were obtained from heart puncture and analyzed for adiponectin, IL-6, TNF-α, and RANKL by using the Luminex^® 200™ Total System (Luminex Corporation, Austin, TX, USA) as previously described [33 (link)].