Chondroitin sulphate from bovine trachea

Sulphated polysaccharide concentration was measured in all samples and pooled fractions using the Blyscan™ Sulphated Glycosaminoglycan assay according to manufacturer’s instructions (Biocolor Ltd., Carrickfergus, County Antrim, UK), with modifications. Blyscan Dye Reagent (250 μL) was added to 25 μL sample, blank or standard (chondroitin sulphate from bovine trachea, Sigma-Aldrich, Castle Hill, NSW, Australia) in triplicate, in a 96 well V-bottom plate (Stor Plate-96 V-bottom, Perkin Elmer, Waltham, MA, USA). The plate was placed on an orbital shaker for 30 min (500 rpm) followed by centrifugation at 3270× g (Beckman Coulter, Allegra™ X-12R, Lane Cove, NSW, Australia) for 10 min. Supernatant was removed without disturbing the pellet using a vacuum before 250 μL Blyscan Dye Dissociation Reagent was added to each well. The plate was again placed on the orbital shaker for 30 min (500 rpm) or until complete dissociation of the pellet. 200 μL of the resuspended solution was transferred from each well into a 96 Well Plate to enable absorption to be measured at 656 nm in a Spectra-Max M3 System spectrophotometer. Sulphated polysaccharides were calculated from the standard curve using SoftMax-Pro 6.1 software.

After 21 days of culture, pellets were digested overnight with 0.5 mg/ml proteinase-K (Sigma-Aldrich) at 56 °C, followed by deactivation at 95 °C for 10 min. DNA content was quantified with Hoechst 33258 (Sigma-Aldrich) using a microplate reader (Victor3 Micro Plate Reader, PerkinElmer, Waltham, MA, USA) with excitation at 360 nm and emission at 465 nm according to published methodology [23 (link)]. In the same samples, quantification of glycosaminoglycans (GAG) was carried out via the 1, 9-Dimethyl-methylene Blue (DMMB) assay [24 (link)], with Chondroitin Sulphate from bovine trachea (Sigma-Aldrich) used as standard. Absorbance at 535 nm was measured.