Gomori staining kit

Cells osteogenically induced for 14 days were washed with precooled PBS for three times before being stained with ARS to examine the matrix concentrations of calcium according to a previously described procedure [3 (link)]. The ALP Quantitative Analysis Kit (Beyotime, Shanghai, China) was used to analyze the ALP activity in cells according to the manufacturer's instructions. Definition of the ALP activity unit is as follows: hydrolysis of para-nitrophenyl (unit: μmol) per minute at 37°C in a diacetamine (DEA) buffer pH 9.8. The absorbance was measured at 405 nm. ALP staining was performed using the Gomori Staining Kit (Nanjing Jiancheng Institute, China). Finally, the samples were washed with deionized water and visualized by phase microscopy using an inverted microscope. ARS reverse quantification was by stain extraction, and the absorbance was measured at 405 nm.

Dulbecco’s modified Eagle’s medium low glucose (DMEM-LG), fetal bovine serum (FBS), trypsin, dexamethasone (Dex), β-glycerophosphate (β-GP) and vitamin C (vit C) were purchased from Gibco (Grand Island, NY). U0126, antibodies specific for ERK1/2, phospho-ERK1/2, phospho-p38-MAPK monoclonal antibody, IgG/HRP and β-actin polyclonal antibody were purchased from Cell Signaling Technology (Beverly, MA). Gomori staining kit, alizarin red dye solution, rat collagen-I (Col-I) and ELISA detection kit were obtained from the Jiancheng Bioengineering Institute (Nanjing, China). TRIzol was purchased from Invitrogen (Carlsbad, CA). Primer synthesis and RT, PCR kits were purchased from Sangon Biotech (Shanghai, China). The CCK-8 kit and Western blotting reagents were purchased from Beyotime Biotechnology Institute (Haimen, China).

The analysis of ALP activity and ALP staining was performed using cells osteogenically induced for 0, 3, 7 and 14 days. ALP activity was analyzed using the ALP Quantitative Analysis Kit (Nanjing Jiancheng Institute, China), and ALP distribution was analyzed using the Gomori Staining Kit (Nanjing Jiancheng Institute, China) according to the manufacturers’ instructions. All experiments were performed in triplicate, and three independent experiments were performed.