Blyscan sgag assay kit b1000

Human NP cells were treated with IL-1β (10 ng/mL) or IL-1β + CoPP (10 μM) for 6 days. Then, the DMMB assay was performed using a Blyscan™ sGAG Assay kit (B1000, Biocolor Ltd., Carrickfergus, UK) according to the manufacturer’s instructions. Briefly, harvested NP cells were washed with PBS then digested with 1mLpapain extraction reagent for 24 hours at 65 °C. The sGAG content was determined by reaction with DMMB, and staining was quantified by measuring absorbance at 656 nm. Chondroitin-4-sulfate was used as the standard.

Cartilage tissues dissected from the developing knee of E18.5 embryos were weighted, snap frozen in liquid N₂ and stored at −80°C until assayed. Tissues were digested in screw cap tubes for 6 h at 60°C with agitation in a 125 μg/mL papain solution containing 0.1 M sodium acetate pH 5.5, 5 mM EDTA, and 5 mM cysteine hydrochloride (all Sigma-Aldrich). Afterwards, samples were cooled down and centrifuged at 10,000 g for 10 min and supernatant collected. Sulphated glycosaminoglycans (sGAG) were quantified via the Blyscan sGAG assay kit (B1000; Biocolor Ltd., Carrickfergus, UK) according to manufacturer`s protocol. Optical density was measured at 650 nm on a Multiscan FC microtiter-plate reader (Thermo-Scientific) and plotted against a chondroitin 4-sulfate standard curve. Results are given as µg of sGAG per mg of wet tissue.