Quantifying Total sGAGs in ECM Using DMMB Assay

Total sGAGs content was quantified with a 1,9-Dimethyl-Methylene Blue zinc chloride double salt (DMMB) assay based on reported protocols [26 (link),33 (link),34 (link)]. For this, 20–25 mg of ECM powder was incubated in 300 µL of 75 mM NaCl, 25 mM EDTA, 50 µL of 10% SDS, and 5 µL of proteinase K (19.9 mg/mL) (Thermo Scientific) at 60 °C overnight. Next, 20 µL of digested organ-derived ECM was mixed with 200 µL of DMMB solution (comprising 19 mg DMMB in 40 mM Glycine, 38 mM NaCl, 100 mM acetic acid pH 3) and the absorbance read at 525 nm immediately. Serial dilutions of chondroitin sulphate (Sigma-Aldrich) were used for the calibration curve, and the absorbance blank corrected with DMMB solution. The total sGAGs content (µg/mL) from each organ-derived ECM was calculated from four independent experiments each performed in triplicate (Figure 1d).