Murine recombinant il 1β

Primary cartilages from femoral head were isolated from 6-weeks-old C57BL/6 mice, washed twice by PBS, placed into 96-well plates (1 in each well) and cultured in 200 μl of pre-warmed medium (DMEM low-glucose supplemented with 10% FBS and 25 mg/L penicillin/streptomycin) for 24 h (Stanton et al., 2011 (link); Marino et al., 2016 (link)). Cartilages were stimulated with 10 ng/ml murine recombinant IL-1β (Biolegend) or recombinant 10 μg/ml FliI (Novus Biologicals) for 72 h. The culture supernatant was harvested for measuring the release of glycosaminoglycan (GAG) using 1,9-Dimethylmethylene blue (DMMB) dye binding assay (Stanton et al., 2011 (link); Terkeltaub et al., 2011 (link)). Cartilages were fixed using 4% PFA (Wako, Japan) for 48 h, decalcified by EDTA solution (Wako) for 2 weeks and then embedded in paraffin. Sections with 5 μm thickness were prepared and stained with hematoxylin and eosin and Safranin O. Safranin O staining indicating pathological changes was quantified using ImageJ (Schneider et al., 2012 (link)) (National Institutes of Health; NIH, USA) in the superficial area of articular cartilage and subtracted from the total area (Headland et al., 2015 ).