2.2.7. Alcian Blue Glycosaminoglycan Assay

The GAG content was measured for samples extracted by the experimental protocols using the Alcian blue GAG assay [25]. To 20 µL of each sample, 0.027 M H₂SO₄, 0.375% Triton X-100, and 4 M guanidine-HCl in a ratio of 1:1 (v/v) was added. GAGs were stained with 0.2 mL of working dye solution containing 0.25% Triton X-100, 0.018M H2SO4, and 0.005% Alcian blue stain. The samples were stirred for 10 min and centrifuged (16,000 rpm, 10 min at 4 °C). The supernatant was discarded, and the stained GAG pellet was mixed with 500 μL of 8 M guanidine-HCl solution. The absorbance of each sample was measured at 600 nm using a Beckman spectrophotometer (DU 640) and compared with a shark cartilage chondroitin sulfate standard curve. The procedure was carried out in duplicate.