Several protocols were combined and modified to generate a DMMB assay for our study [60 (link),61 (link),62 (link),63 (link)]. A mixture was prepared by dissolving 21 mg of DMMB (Sigma, 341088) in 5 mL of absolute ethanol, followed by the addition of 2 g of sodium formate (Sigma). Then, the solution was mixed with 800 mL of double distilled water. Formic acid (95%, Sigma) was added dropwise to the solution until pH was 1.5, and then, distilled water was added to make 1L of final solution.
The process involved transferring 50 µL of centrifuged and digested samples into a 96-well plate. To this, 250 µL of DMMB was added to each well, and the contents were mixed by pipetting up and down. After incubation at room temperature for 1 h, a spectrophotometric plate reader (Thermo Fisher) was used to measure optical density (OD) of each well at 525 nm. A linear standard curve was also generated using various known concentrations of type A chondroitin sulfate (from bovine trachea, Sigma-Aldrich), and the GAG content of the hydrogel samples was extrapolated from the standard curve.
The process involved transferring 50 µL of centrifuged and digested samples into a 96-well plate. To this, 250 µL of DMMB was added to each well, and the contents were mixed by pipetting up and down. After incubation at room temperature for 1 h, a spectrophotometric plate reader (Thermo Fisher) was used to measure optical density (OD) of each well at 525 nm. A linear standard curve was also generated using various known concentrations of type A chondroitin sulfate (from bovine trachea, Sigma-Aldrich), and the GAG content of the hydrogel samples was extrapolated from the standard curve.
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