Blyscan sulfated gag assay kit

Tissue pieces (~ 30–40 mg) were obtained by punch biopsy tool and dried in 60 °C oven overnight. Dried tissue pieces were digested in Papain solution at 65 °C for 18 h. Corresponding native flap tissues were dried and digested in parallel as controls. Papain (Sigma Aldrich,  ≥ 16 units/mg protein) 15–30 mg/mL stock was solubilized to working concentration of 0.1 mg/ml in 0.1 M phosphate buffer (pH 6.0), with 5 mM cysteine hydrochloride (Sigma Aldrich), and 5 mM EDTA (Sigma Aldrich). The lysates were used for detection of sulfated glycosaminoglycan (sGAG) and DNA content. The Blyscan Sulfated GAG Assay kit (Biocolor) was used to measure sGAG according to manufacturer’s instruction. Briefly, tissue specimen lysates were mixed with Blyscan Dye Reagent to bind the GAG for 1 h at room temperature. The GAG-dye complex was then collected by centrifugation at 10,000×g. After the supernatant was removed and the tube drained, Dissociation Reagent was added and 100 μl of analyte solution was transferred to a 96-well plate. Absorbance against the background control was obtained at a wavelength of 656 nm with a SpectraMax spectrophotometer (Molecular Devices). GAG amount was interpolated from a standard curve (0–5 µg) using a known GAG standard provided in the kit. Final GAG content was standardized to the total dry tissue mass (mg) used for assay.

Total carbohydrates content was determined by anthrone method, using a standard curve of glucose in the range of concentrations 0–100 μg/mL [27 ]. The hexosamine content was determined using the Ehrlich reagent method [28 (link)]. A standard curve was built using glucosamine hydrochloride in the range of concentrations 0.03–0.15 mM. Determination of uronic acids content was performed using the orcinol-based method, as previously described [29 (link)]. The standard curve was built using glucuronic acid in the range of concentrations 0.03–0.15 mM. Total protein content was determined using the Biuret method and bovine serum albumin as a standard in the range of concentrations 0–10 mg/mL. Sulfate content was estimated by barium chloride assay [30 ], using potassium sulfate in the range of 0.25–4 mg/mL to build the standard curve. All spectrophotometric analyses were carried out at a V-650 UV-VIS spectrophotometer (Jasco, Japan). The content of O- and N-sulfated GAGs and their ratio were determined using Blyscan sulfated GAG assay kit (Biocolor, UK), according to the manufacturer’s instructions. CS sodium salt from bovine trachea was used to build the standard curve. The absorbance was read at a SPECTROstar nano microplate reader (BMG Labtech, Germany). A number of six measurements was taken for each chemical analysis.

The sGAG content in the DEWJ was measured using a Blyscan sulfated GAG assay kit (Biocolor, UK) according to the manufacturer’s instructions. Briefly, to extract sGAG, the samples were digested with a papain solution (with a concentration of 125 mg mL⁻¹ papain in GAG buffer) (Sigma-Aldrich, USA) and placed in a water bath at 60 °C overnight. The suspension was centrifuged at 10,000g for 10 min. About 100 µL of the supernatant containing sGAG was mixed with 1 mL Blyscan dye and shaken for 30 min. The precipitate was collected by centrifugation at 12,000 rpm for 10 min and then dissolved in 1 mL of dissociation reagent. The absorbance was measured in a 96-well plate at 656 nm using a multiplate reader (H4, BIO-TEK Instruments Inc., USA).

To verify the extent of decellularization, histological analyses and biochemical assays were performed to evaluate the content of DNA, sGAG and collagen in the decellularized tissue. For histological evaluation, both native and decellularized tissues were fixed in paraformaldehyde, embedded in paraffin wax, sectioned at 6 ​μm with a microtome (Leica, Germany). The sections were then stained with alcian blue to access sGAG content and picro-sirius red for collagen.
For biochemical analysis, the dECM samples and native tissue were enzymatically digested with papain solution (125 ​μg/mL papain in 0.1 ​M sodium phosphate with 5 ​mM Na₂-EDTA and 5 ​mM cysteine-HCl at pH 6.5) for 16 ​h at 60 ​°C. DNA content was quantified using a Quant-iT Pico Green dsDNA assay kit (Invitrogen) according to the manufacturer's protocol. Quantification of sGAG in the digested samples were performed using a dimethylmethylene blue (DMMB) assay (Blyscan sulfated GAG assay kit, Biocolor). Collagen content was indirectly quantified by measuring hydroxyproline content using a Chloramine-T assay, assuming a hydroxyproline to collagen ratio of 1:7.69 [41 ].