Quantification of Sulfated Proteoglycans and GAGs

Sulfated proteoglycans and GAGs were quantified using the 1,9-dimethylmethylene blue (DMMB; Sigma-Aldrich, MA, USA) protocol [82 (link)]. The iPSCs and neurons were digested in a prepared papain solution (containing 1 mM EDTA, 2 mM DTT, and 0.3 mg/mL papain enzyme) (Sigma-Aldrich, MA, USA) at 60 °C for 1 h. Iodoacetic acid (Sigma-Aldrich, MA, USA) was then added to a final concentration of 10 mM, followed by the addition of 0.5 ml of 50 mM Tris/HCl (Sigma-Aldrich, MA, USA). Briefly, 20 μL of sample was mixed with 200 μL of DMMB reagent, and absorbance was read at 525 nm. A standard curve was established from chondroitin-6-sulfate (Sigma-Aldrich, MA, USA) to compare absorbance for the samples. The DNA content was measured using a fluorometric assay. Total GAG levels were normalized to total DNA content. Aliquots of the sample digestion were stained with 200 μL of Hoechst33258 (Cayman Chemical, MI, USA) working solution (2 μg/mL). The fluorescence intensities were then detected at 355 nm for excitation and 460 nm for emission. Both Hoechst-stained DNA and GAG contents were measured by a SpectraMax i3x Multi-Mode Microplate reader (Molecular Devices, CA, USA).