1,9-dimethylmethylene blue (DMMB) colorimetric assay for sulfated GAGs

NP tissue was isolated separately from five lumbar and ten tail IVDs of each sample from both NOD and control mice and digested using papain at 60°C for 2 hours. GAG content was measured in duplicates by DMMB procedure¹¹ using chondroitin-6-sulfate (C-8529, Sigma, Milwaukee, WI) as a standard. The DNA concentration of each sample was measured using the PicoGreen^® assay (P-11496, Molecular Probes, Sunnyvale, CA) to normalize GAG values.