Blyscan sulphated glycosaminoglycan assay

Protein levels of TIMP-1, TIMP-2, MMP-1, MMP-3, MMP-13, MMP-2, MMP-9, IL-1β, TGFβ3, TGFβ2, thrombospondin-1 (R&D Systems), TIMP-3, VCAM-1 (Boster Biological Technology), TGFβ1 (IBL International), MIA (Roche), IL-8 (Invitrogen), and aggrecan (DIAsource ImmunoAssays S.A.) in supernatants were measured on days 3 and 7 by commercially available ELISA kits according to the manufacturer's instructions. IL-6 was measured by flow cytometry using Diaclone DIAplex IL-6 kit (Gen-Probe), and glycosaminoglycans were measured by blyscan-sulphated glycosaminoglycan assay (Biocolor). ELISA data were normalized by subtracting the values of medium from the samples.

Dot blot: Serum-free conditioned medium (DMEM) from SH-SY5Y cells, cultured for 72h, was spotted onto nitrocellulose membrane using a dot blot apparatus. CSPG mix (Millipore) diluted with DMEM were used as a positive control. Half the samples from each group were treated with ChABC for 3h, prior to incubation of the membrane with antibody 2B6 (mouse monoclonal to epitopes exposed by ChABC digestion) 1:500, Seikagaku. This detects a stub epitope exposed by ChABC digestion of CSPGs. The membrane was then washed and incubated with HRP anti-mouse IgG 1:10,000. The signal was detected using chemiluminescence (Luminata) and visualised using hyperfilm (Amersham).
CSPG assay: A Blyscan sulphated glycosaminoglycan assay (Biocolor) [24 (link)], was used to detect the presence of glycosaminoglycan chains in the conditioned medium of differentiated SH-SY5Y cells. This dye binding assay is a quantitative measure of intact CSPG GAG chains. A CSPG mix (Millipore) was used as a positive control. Absorbance was measured at 656nm and sulphated-glycosaminoglycan concentrations obtained from a standard curve.

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.