Papain suspension

Sulfated glycosaminoglycan (sGAG) was quantitatively determined using Glycosaminoglycans Assay Kit (Chondrex, Inc., Washington). sGAG were extracted from the dECM samples using papain extraction reagent. To create the papain extraction solution, 0.1M sodium acetate, 0.01M Na₂ETDA (EMD Millipore Corporation, USA), and 0.005M cysteine hydrochloride were added to 0.2M sodium phosphate buffer (sodium phosphate monobasic and sodium phosphate dibasic). Once all the components were completely dissolved, papain suspension (Sigma-Aldrich, USA) was added to the extraction buffer and stored at 4°C for a maximum of 10 days. Papain extraction solution was then added to the dECM samples and incubated at 37°C for 3 h. Digested aliquots were collected from the wells and mixed with 1,9 Dimethylmethlyene (DMB) Dye Solution as per manufacturer’s instructions. The concentration of GAGs in samples were determined using the Eon plate spectrophotometer and regression analysis.

Rat tail high concentration collagen type I was obtained from Corning (Bedford, MA). Sodium hydroxide, fluorescein isothiocyanate-dextran of 150K MW (FITC-dextran), sodium acetate, cysteine hydrochloride, sodium phosphate monobasic, sodium phosphate dibasic, papain suspension, cytochalasin D, and (–)-blebbistatin were procured from Sigma Aldrich (St. Louis, MO). Y-27632 and Na₂EDTA were purchased from EMD Millipore (Kankakee, IL). Dulbecco’s Phosphate Buffer Saline (DPBS), RPMI Medium 1640, Penicillin-Streptomycin (Pen Strep), 0.25% Trypsin-EDTA, fetal bovine serum (FBS), and collagenase Type I were acquired from Gibco by Life Technologies (Grand Island, NY). D-(–)-Ribose, Hoechst 33342, and Alexa Fluor™ 488 phalloidin were obtained from Thermofisher Scientific (Waltham, MA). AlamarBlue™ cell viability reagent was purchased from Invitrogen by Thermo Fisher Scientific (Eugene, OR). Acetone, methanol, and glacial acetic acid were acquired from Fisher Chemical (New Jersey). Endothelial cell basal medium-2 (EBM-2) and EGM-2 SingleQuots growth factors were obtained from Lonza (Walkersville, MD).