Blyscan glycosaminoglycan kit

To prepare the samples for dsDNA and glycosaminoglycan (GAG) quantification, 50 mg of lyophilized liver dECM or native liver control was measured and placed into a 1.5 mL eppendorf tube. Then 1 mL of papain digest solution composed of 0.1 mg/mL papain (Sigma-Aldrich) in 0.2 M sodium phosphate buffer solution (pH 6.4) was added to each sample. The samples were then digested at 65°C for 20 h with periodic vortexing, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was collected and used to measure the residual dsDNA content with the Quant-iT^™ PicoGreen dsDNA (ThermoFisher Scientific) and the GAG content with the Blyscan^™ Glycosaminoglycan Kit (Biocolor), respectively, according to the manufacturer’s protocols.
To quantifying the residual collagen content, 10 mg of lyophilized liver dECM or native liver control was measured and placed into a 1.5 mL polypropylene tube. Next, 100 µL of deionized water was added and vortexed on high for 30 s to homogenize the sample followed by the addition of 100 µ L of 12 M HCl (MilliporeSigma). The samples were vortexed briefly and hydrolyzed for 3 h at 120°C followed by centrifugation at 10,000 rpm for 10 min. The supernatant was collected and used to quantify the collagen content using the Hydroxyproline Assay Kit (Sigma-Aldrich) according the manufacturer’s methods.

Decellularized tissue samples as well as their respective native tissue controls were frozen and lyophilized for 48 h. For dsDNA and glycosaminoglycan (GAG) content measurement, 50 mg of dried powdered sample was placed in a 1.5 mL eppendorf tube and digested with 1 mL of papain solution (Cat. # P3125, Sigma-Aldrich) comprised of 0.1 mg/mL papain in 0.2 M sodium phosphate buffer solution (pH 6.4) at 65°C for 20 h with periodic vortexing. After digestion, the samples were centrifuged at 10,000 rpm for 10 min and the supernatant was collected. Residual dsDNA content was measured using the Quant-iT™ PicoGreen dsDNA (ThermoFisher Scientific) and the GAG content with the Blyscan™ Glycosaminoglycan Kit (Biocolor) following the manufacturer’s instructions. To quantify the collagen content, 10 mg of dried powdered sample was placed in a 1.5 mL polypropylene tube and homogenized with 100 μL of DI water for 30 s using a vortex. Next, 100 μL of 12 M HCl (Millipore Sigma) was added to each tube and hydrolyzed at 120°C for 3 h in a heat block. The samples were centrifuged at 10,000 rpm for 10 min and the supernatant was collected for collagen measurement using the Hydroxyproline Assay Kit (Sigma-Aldrich) according the manufacturer’s protocol.