Quantifying Sulfated Glycosaminoglycan Production

On day 6, one half of each agarose construct was used to examine for PG content. Each sample was placed in 500 µL papain buffer (pH 7.4) containing 250 µg/ml papain (Sigma-Aldrich, St. Louis, Mo), 100 mM sodium phosphate, 10 mM EDTA and 10 mM cysteine HCl and incubated at 60 °C for 16 h. The amount of sulfated glycosaminoglycan in the digestion solution was determined by Dimethylmethylene (DMMB) blue dye-binding assay. Briefly, 20 µL aliquot of the papain digest was mixed with 200 µL of DMMB staining solution (pH = 1.5)⁸⁵ . Absorbance of the mixture was read at 525 nm by the multimode plate reader. Chondroitin sulfate from bovine trachea (Sigma-Aldrich, St. Louis, Mo) dissolved in the same papain buffer was used as the standard. All measurements were normalized by DNA content as determined by the Hoechst 33258 fluorometric assay^{86 (link)}. The PG production was defined as an increased amount of PG content over 6 days by comparing with the PG content of agarose constructs on day 0.