Dimethyl methylene blue chloride

To quantify the content of GAG in the samples, the reaction of GAG with dimethylmethylene (DMMB) blue was measured. This technique relies on the absorption spectra of the change in 1,9-dimethylmethylene dye due to the induction of metachromasia when it binds to sulfated GAGs.⁵¹ (link)
Frozen cartilage sections were digested overnight at 56 °C in 500 μl 1 mg/ml proteinase-K solution in digestion buffer (Tris/EDTA buffer). dimethylmethylene blue chloride (16 μg/ml dimethylmethylene blue) (Sigma-Aldrich, USA) in a solution of 3.04 mg/ml glycine and 2.37 mg/ml NaCl dissolved in 0.01M HCl in d₂H₂O (pH 3) was added directly followed by the measurement of the absorbance at 520 nm using a Varioskan Flash Multimode Reader (Thermo Scientific). Chondroitin sulphate-B (Sigma-Aldrich, USA) was used to generate a standard curve, and GAG measurements were normalized to the wet weight.⁵² (link) An ANOVA with a selected significance threshold of p < 0.05 was used to determine statistically significant differences between the GAG content for each layer of cartilage.

The GAG production was analyzed using the dimethylmethylene blue (DMMB) GAG assay. At each time point, the same digested samples used in the DNA assay were used for the DMMB assay to quantify GAG/DNA. Briefly, 1,9–dimethylmethylene blue chloride (Sigma) was dissolved in ethanol overnight and then added to a sodium chloride (0.04 M NaCl)/glycine solution, pH 3, for a final concentration of 46 μM DMMB. The solution was filtered, and the absorbance at 525 nm was measured to be 0.314 OD. Next, a serial dilution of chondroitin sulfate (CS; Sigma), ranging from 0 to 100 µg/ml, was prepared using PBE. Finally, 30 μl of the samples or the CS standards was loaded into a 96-well plate, along with 270 μl of the DMMB dye solution, and the absorbance was read at 570 nm. The GAG concentration was calculated using the standard curve generated from the serial dilution of CS, and then divided by its corresponding DNA content to calculate GAG/DNA (µg/pg).

Glycosaminoglycan (GAG) content of vitro‐EC in different groups (n = 6 in each group) was quantified by dimethyl methylene blue chloride (Sigma‐Aldrich) [23]. Total GAG was precipitated by guanidinium chloride solution (0.98 mol/L). After the GAG precipitate was dissolved, the optical density (OD) was determined at 595 nm. A standard curve was established according to the OD values of chondroitin‐4‐sulfate with different concentrations. The total GAG amounts were determined on the basis of the OD value and the standard curve.