Sulfated glycosaminoglycan (sGAG) assay

Pellet cultures were digested overnight at 60 °C with 300 μg/ml papain (Millipore Sigma, St. Louis, MO, USA) in 20 mM sodium phosphate buffer (pH 6.8) containing 5 mM EDTA and 2 mM dithiothreitol (DTT). Cell lysates were clarified by centrifugation and sGAG was determined using the Blyscan™ sGAG assay kit (Biocolor Ltd, Carrickfergus, UK) according to the manufacturer’s protocol. Briefly, cell lysates were incubated with the 1,9-dimethylmethylene blue (DMMB) dye reagent for 30 min and unbound dye was removed by centrifugation. The bound dye was dissociated from the sGAG–dye complex and quantified by spectrophotometry based on A656. Using chondroitin 4-sulfate as a standard, total sGAG was determined and expressed as a function of the protein content.