Enzyme-linked immunosorbent assay (ELISA) of proinflammatory cytokines

RAW264.7 cells were cultured at 1 × 10⁵/well in 48-well plates (SPL life sciences Co., Pocheon, Korea) for 24 h. The cells were washed with fresh medium and treated with ChondroT or GHJTY (1, 0.3, or 0.1 mg/mL) for 2 h, followed by treatment with 500 ng/mL LPS (Sigma Co., MO, USA) for 24 h. IL-6 (Biolegend, San Diego, CA, USA), TNF-α (R&D system, Minneapolis, MN, USA), IL-1β (R&D System, Minneapolis, MN, USA), and PGE₂ (R&D system, Minneapolis, MN, USA) in the supernatants were measured using ELISA kits following the manufacturer’s experimental protocols. The assay was performed at room temperature and the optical absorbance was measured at 450 nm using an ELISA microplate reader (ELx808) within 30 min.