B1000

Cultured samples were washed with Dulbecco’s phosphate-buffered saline (DPBS; DSBN200, DS-Pharma, Osaka, Japan) and digested at 65°C with 0.5 mg/ml papain (P3125, Sigma-Aldrich) dissolved overnight in a 0.2 M sodium phosphate buffer solution supplemented with 8 mg/ml sodium acetate (#192–01075 Wako Pure Chemical), 4 mg/ml ethylenediaminetetraacetic acid sodium salt dihydrate (E-5134, Sigma-Aldrich) and 0.8 mg/ml L-cysteine (C-7880, Sigma-Aldrich). Sulfated GAG content was measured with a 1,9-dimethyl-methylene blue (DMMB) assay (Blyscan; B1000, Biocolor, Belfast, UK) with chondroitin-6-sulfate (B1000, Biocolor) as the standard, using a spectrophotometer to measure absorbance at 656 nm. DNA quantity was determined using a PicoGreen assay (P11496, Thermo Fisher Scientific) with a spectrophotometer in fluorescence mode with excitation at 480 nm and emission at 520 nm. Each colorimetric measurement was done for three wells in 96 well plates for each group of samples from five donors. NTP concentrations of 0.1 and 1.0 mNU/ml were used.

The DMMB (1,9‐dimethylmethylene blue) assay was performed using a Blyscan sGAG assay kit (Biocolor, B1000, Biocolor, Carrickfergus, UK) according to the manufacturer's instructions. Briefly, harvested NP tissues or NP cells were washed with PBS and digested with 1 mL papain extraction reagent for 24 h at 65 °C. CS contents were determined by reaction with DMMB, and staining was quantified by measuring absorbance at 656 nm. Chondroitin‐4‐sulfate was used as the standard.

Lyophilized samples (3–5 mg) from myocardium, aortic wall and cusps (n = 6) were used for glycosaminoglycans quantification, according to the manufacturer’s instructions (Blyscan Sulfated Glycosaminoglycan Assay, Biocolor, B1000, County Antrim, UK). This assay is based on the protocol established in [18 (link)]; the end point absorbance (656 nm) was measured spectrophotometrically.