Radioimmunoprecipitation assay (ripa)

The quantification of the GAG content was conducted by a modification of the dimethylmethylene blue method (Beyotime Institute of Biotechnology) (14 (link)). The chondrocytes were treated as follows: the cells were seeded in 6-well cell culture plates at a density of 1×10⁵ cells/well and incubated for 24 h to allow attachment. The medium was changed with 3 ml of extraction medium every 2 days. Following incubation of the cells under the cell culture conditions for 5 days, the 6-well cell culture plates were observed under an optical microscope. Three parallel wells were established for each treatment condition. After rinsing in PBS (pH 7.4; Gibco-BRL), a cytolysate (RIPA; Wuhan Boster Biological Technology, Ltd.) was added to each well at 0.4 ml/well and vortexed for 10 sec. An aliquot (40 µl) of the digest was assayed for the total GAG content by the addition of 200 µl of 1,9-dimethylmethylene blue dye solution (Beyotime Institute of Biotechnology). The absorbance was determined at 595 nm using a microplate reader (Bio-Rad 680; Bio-Rad). The amount of GAG was extrapolated from a standard curve based on shark chondroitin sulfate.

The chondrocytes were treated as described above. The cells were seeded in 12-well plates at a density of 5×10⁴ cells/well. The cells were then incubated for 24 h to allow attachment. The medium was then changed with 2 ml of extraction medium every 2 days. Following incubation of the cells in a humidified atmosphere (95%) with 5% CO₂ at 37°C for 5 days, the 12-well cell culture plates were observed under an optical microscope. Three parallel wells were established for each treatment condition. After the cell culture plates were placed on ice and rinsed in PBS (pH 7.4; Gibco-BRL), a cytolysate (RIPA; Wuhan Boster Biological Technology, Ltd.) was added to each well at 0.2 ml/well, vortexed for 10 sec, and centrifuged for 10 min at 10,000 x g according to the manufacturer’s instructions. An ELISA kit (R&D Systems, Minneapolis, MN, USA) was used for the quantitative determination of rabbit Col II levels in the cell cultures from each well. ELISA was conducted according to the manufacturer’s instructions. The spectrophotometric absorbance of the samples at 450 nm was measured using a microplate reader (Bio-Rad 680; Bio-Rad). The standard curve was calculated according to the concentration and absorbance of the standard, asw ell as based on the curve to extrapolate the corresponding concentration of the sample.