1 9 dimethylmethylene blue dmb

Sodium alginate was purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). Chitosan was obtained from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Cysteine, hematoxylin and eosin (H&E) staining kit, papain, and safranin-O were kindly purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Masson stain kit was obtained from Jiancheng Technology Co., Ltd. (Nanjing, China) C5.18 cells, acridine orange/ethidium bromide (AO/EB) and klcian blue staining Kit were provided by Syagen Biosciences Inc. (Guangzhou, China). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 1,9-dimethylmethylene blue (DMB) was obtained from Sigma Co., Ltd. (St. Louis, MO, USA). Rat collagen type-II (Col-II) ELISA kit was purchased from Cusabio Biotech Co., Ltd. (Wuhan, China). Basic fibroblast growth factor (bFGF) was purchased from PeproTech Inc. (Rocky Hill, CT, USA).

The pellets were digested using 1 mg/mL Proteinase K, 1 mM iodoacetamide, 10 µg/mL Pepstatin A in 50 mM Tris, 1 mM EDTA buffer (250 μL) (pH 7.6; all Sigma-Aldrich, St. Louis, MO, USA) for 16 h at 56 °C, followed by Proteinase K inactivation at 100 °C for 10 min. To determine the amount of DNA, the cell lysates were treated with 0.415 IU/mL heparin and 1.25 µg/mL RNase for 30 min at 37 °C, followed by addition of 0.375 µL CYQUANT GR solution (ThermoFisher, Waltham, MA, USA). The samples were analysed using a SpectraMax Gemini plate reader with an excitation of 480 nm and an emission of 520 nm. As a standard, DNA sodium salt from calf thymus (Sigma-Aldrich, St. Louis, MO, USA) was used. To determine the amount of GAG, the cell lysates were diluted in PBS supplemented with 10 mM EDTA (pH 6.5) to a volume of 50µL and mixed with 200 µL of 32 mg/L 1,9-dimethylmethylene blue (DMB, Sigma-Aldrich, St. Louis, MO, USA) in 0.04 M Glycin, 0.04 M NaCl pH 3.0. Then the absorbance was measured on a Versamax microplate reader at 590 nm and 530 nm. A 530:590 nm ratio was used to determine the glycosaminoglycan concentration. As a standard, chondroitin sulphate sodium salt from shark cartilage (Sigma-Aldrich, St. Louis, MO, USA) was used.