Penicillin streptomycin

Colistin (C-4461), lipopolysaccharides (LPS) (L4391), and carboxylate-modified fluorescent microspheres (L4655, 1.0 µm in diameter) were purchased from Sigma, USA. p38 inhibitor (SB203580) was purchased from MedChemExpress (USA). Fetal bovine serum (FBS) was obtained from Gibco (USA), and bovine serum albumin (BSA) was supplied by Roche (Switzerland). RPMI-1640 medium and phosphate buffered saline (PBS) were provided by HyClone (USA). DMSO and thiazolyl blue (MTT) were purchased from AMRESCO (USA); trypsin and penicillin-streptomycin were obtained from TBD Science (China). Phospho-p38 MAP kinase (Thr180/Tyr182) antibody (#9211) was supplied by Cell Signaling (USA). Horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody was provided by Solarbio (China), and antibody against β-actin was obtained from Thermo Fisher (USA). Enzyme-linked immunosorbent assay (ELISA) kits for various cytokines were purchased from R&D Systems (USA).

Primary chondrocytes were harvested according to a previous protocol.24 The New Zealand rabbits were purchased from the Third Military Medical University (Chongqing, China). The experiment was approved by Animal Care and Use Committee of the Third Military Medical University. Articular cartilage was removed from the knee joint of rabbits (1 month old, n = 3) with a sterile blade and minced. Cartilage pieces were digested with 0.2% w/v type collagenase (Sigma, St. Louis, MO, USA) in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 1% v/v penicillin‐streptomycin (Gibco) at 37°C in a 5% CO₂ incubator overnight. Digested tissue was filtered with a 40‐μm‐cell strainer (BD Bioscience, Franklin Lakes, NJ, USA) followed by centrifugation at 400 g for 5 minutes and resuspended in DMEM/F12 supplemented with 10% v/v foetal bovine serum (Tbdscience, Tianjin, China) and 1% v/v penicillin‐streptomycin. Medium was changed every 3 days. Cells were passaged at 80%‐90% confluence, and the passage 2 (P2) cells were used in the following experiments. For human TGF‐β1 stimulation, chondrocytes were treated with 5 ng/mL TGF‐β1 (Peprotech, Rocky Hill, NJ, USA) for 24 hours.