Blyscan sulfated glycosaminoglycan sgag assay kit

Samples harvested were digested with 10 mg/mL of pepsin in 0.05 M acetic acid at 4 °C, followed by digestion with elastase (1 mg/mL). A Blyscan sulfated glycosaminoglycan (sGAG) assay kit (Biocolor Ltd., Newtownabbey, Ireland) was used to quantify sGAG deposition according to manufacturer’s protocol. Absorbance was measured at 656 nm, and sGAG concentration was extrapolated from a standard curve generated using a sGAG standard. Type II Collagen (Col 2) content was measured using a captured enzyme-linked immunosorbent assay (Chondrex, Redmond, WA). Absorbance at 490 nm was measured and the concentration of Col 2 was extrapolated from a standard curve generated using a Col 2 standard. Values for sGAG and Col 2 content obtained were normalized to the total DNA content of respective samples, measured using Picogreen dsDNA assay (Molecular Probes, OR, USA). Quadruplicates of each group were analyzed from two independent experiments.

Samples harvested were digested with 10 mg/mL of pepsin in 0.05 M acetic acid at 4 °C, followed by digestion with elastase (1 mg/mL). A Blyscan sulfated glycosaminoglycan (sGAG) assay kit (Biocolor Ltd., Newtownabbey, Ireland) was used to quantify sGAG deposition according to manufacturer’s protocol. Absorbance was measured at 656 nm and sGAG concentration was extrapolated from a standard curve generated using a sGAG standard. Type II Collagen (Col 2) content was measured using a captured enzyme-linked immunosorbent assay (Chondrex, Redmond, WA). Absorbance at 490 nm was measured and the concentration of Col 2 was extrapolated from a standard curve generated using a Col 2 standard. Values for sGAG and Col 2 content obtained were normalized to the total DNA content of respective samples, measured using Picogreen dsDNA assay (Molecular Probes, OR, USA). Quadruplicates of each group were analyzed from two independent experiments.

All analyses were conducted in triplicate in a blinded manner. The quantification of glycosaminoglycan content involved assessing 100 μL aliquots of tissue homogenates and urine using the Blyscan–sulfated glycosaminoglycan (sGAG) assay kit (Biocolor Ltd., Carrickfergus, UK), following the manufacturer’s protocol. An aqueous solution of 10 mg/mL chondroitin sulfate (Sigma-Aldrich) was used as the reference standard. Absorbance was measured at 660 nm using a Victor3 1420 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). Tissue GAG content was determined from the calibration curve (prepared with chondroitin sulfate amounts ranging from 0 to 5.0 μg in increments of 0.5–1.0 μg) and normalized for DNA concentration, measured using the Quanti-iT PicoGreen dsDNA Reagent (Thermo Fisher Scientific, Waltham, MA, USA) for tissue homogenates, or creatinine concentration, measured using the QuantiChrom Creatinine Assay Kit (BioAssay Systems, Hayward, CA, USA) for urine samples.