3.5. Quantitative evaluation of bovine IVD glycosaminoglycan content

Aliquots of biopsy sample digests described above for PicoGreen analysis were evaluated via dimethylmethylene blue (DMMB) assay. Briefly, fresh control and decellularized NP samples were diluted 1000X and 200X in PBE buffer respectively. Additionally, fresh control and decellularized AF samples were diluted 200x and 100x, respectively. Fifty microliters of each diluted sample were assed in duplicate with 200ul of DMMB reagent (40 mM sodium chloride, 40 mM glycine 46 μM DMMB; pH: 3.0) in a 96-well plate. Absorbance was read at a wavelength of 525nm and GAG content was determined from a standard curve containing known concentrations of chondroitin-6-sulfate.