Sircol gag assay kit

The tissue-engineered constructs (n=4 of each type) were cut into 2.5 mm × 2.5 mm sections and weighted before protein extraction. For collagen extraction, the samples were placed in 1 mL of collagen extraction solution (0.5 M Acetic Acid + 1 U/mL pepsin in water) overnight in an orbital rocker at 20°C. Then, a Sircol insoluble collagen assay kit (Biocolor Ltd., United Kingdom) was used to quantify the amount of collagen in the tissue-engineered constructs following the protocols provided by the company. For GAG extraction, the samples were placed in 1 mL of GAG extraction solution (4 M Guanidine-HCL and 0.5 M Sodium Acetate in water) overnight at 8°C. A Sircol GAG assay kit (Biocolor Ltd., United Kingdom) was used to quantify the amount of GAG in the tissue-engineered constructs following the protocol provided by the company. The collagen and GAG concentrations were determined using an Epoch microplate spectrophotometer (BioTek, USA) set at 550 nm and 656 nm wavelengths, respectively.

Protein extraction was conducted using 2.5 mm x 2.5 mm sections of the cell-cultured constructs. For collagen extraction, the sections were weighed and placed in 1 mL of collagen extraction solution (0.5 M Acetic Acid + 100 U/mL pepsin in water) overnight in an orbital rocker at 20°C. The amount of collagen in the cell-cultured constructs was quantified using a Sircol insoluble collagen assay kit (Biocolor Ltd., United Kingdom) and the protocols provided by the company. The collagen concentration was determined using an Epoch microplate spectrophotometer (BioTek, USA) measuring at 550 nm wavelength. For GAG extraction, the samples were placed in 1 mL of GAG extraction solution (4 M Guanidine-HCL and 0.5 M Sodium Acetate in water) overnight at 8°C. The amount of GAG in the cell-cultured constructs was quantified using a Sircol GAG assay kit (Biocolor Ltd., United Kingdom) and the protocols provided by the company. The GAG concentration was determined using the spectrophotometer measuring at 656 nm wavelength. The measured collagen and GAG concentrations were normalized using the original weights of the cell-cultured constructs, as described in a published protocol [16 (link)].