Il 1β enzyme linked immunosorbent assay kit

Renal tissues were homogenized in ice-cold sucrose buffer (pH 7.4) according to the instructions of the assay kit. A marker of lipid peroxidation, malondialdehyde (MDA), and a marker of oxidative stress and inflammation, myeloperoxidase (MPO) were analyzed according to the protocols of commercial assay kits from Jiancheng Bioengineering Institute (Nanjing, China). The activity of the antioxidant, superoxide dismutase (SOD) was measured using a SOD assay kit from Dojindo Laboratories (Kumamoto, Japan). The level of the pro-inflammatory cytokine IL-1β was measured using an IL-1β enzyme-linked immunosorbent assay kit from R&D Systems (Minneapolis, MN). The values were normalized by tissue protein concentration.

C28/I2 chondrocytes (1 × 10⁵ cells/well) were inoculated in a 6-well plate and processed with indicated treatment for 48 h. Concentrations of Interleukin-1β (IL-1β) in cell culture supernatant were measured by IL-1β enzyme-linked immunosorbent assay kit (R&D Systems, MN, USA). Concentrations of Interleukin-18 (IL-18) and HMGB1 in cell culture supernatant were measured by using IL-18 and HMGB1 enzyme-linked immunosorbent assay kits (Dakewe Biotech, Beijing, China) following the manufacturer’s instructions.

Cell-free supernatants were collected from treated cells by centrifugation at 1500 rpm for 5 min. They were then concentrated using a VIVASPIN 500 (Sartorius, Goettingen, Germany). Protein levels of secreted IL-1β into the culture medium were determined using IL-1β enzyme-linked immunosorbent assay kits (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.