ELISA to measure cytokine levels

The generation of inflammatory gene products (IL-1β, IL-8 and TNFα) by RAW 264.7 cells was assayed using the following ELISA kits purchased from Abcam: Human IL-1β ELISA kit (cat. no. ab46052), human IL-8 ELISA kit (cat. no. ab46032) and human TNFα ELISA kit (cat. no. ab181421). Generation of inflammatory gene products (IL-1β, IL-8 and TNFα) by THP-1-derived macrophages was assayed using the following ELISA kits, mouse IL-1β ELISA kit (cat. no. ab197742; Abcam), mouse IL-8 ELISA kit (cat. no. MOFI01258; ELISA Genie) and mouse TNFα ELISA kit (cat. no. ab100747; Abcam). Cells (1.5×10⁶ cells/well) seeded in 6-well plates were pre-incubated with various concentrations of morin (Mingxiu Biotechnology Company; 100–300 µM) or colchicine (Mingxiu Biotechnology Company; 1 µM) for 24 h at 37°C, and cytokine production was stimulated by treating cells with MSU crystals (1 mg/ml) for 24 h at 37°C. Then, the culture supernatants were collected and used to measure levels of IL-1β, IL-8 and TNFα. Absorbance was determined at 450 nm using a microplate reader (BioTek Instruments, Inc.). The standard curves for respective cytokines were used to quantify the levels of IL-1β, IL-8 and TNFα released by the cells.