Alizarin red s staining solution

AG (chemical formula: C₂₁H₂₀O₁₁, molecular weight: 448.38, purity ≥ 98%) was obtained from Beijing Bailingwei Technology Co. Ltd. (Beijing, China). AG was first dissolved in dimethyl sulphoxide (DMSO), followed by dilution with α-Minimum Essential Medium (α-MEM) to achieve the appropriate concentrations. The final concentration of DMSO in the complete culture medium was no >0.1%. Fetal bovine serum (FBS) and α-MEM were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Alizarin red S staining solution, 4% paraformaldehyde, penicillin and streptomycin, and Trizol reagent were purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China). The alkaline phosphatase assay kit used in this study was obtained from Beyotime Biotechnology Co. Ltd. (Guangzhou, China). Unless otherwise indicated, all other chemical reagents were obtained from Sigma (St. Louis, MO, USA).

MC3T3-E1 cells originated from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). Minimum essential medium-alpha modification (α-MEM) and foetal bovine serum (FES) were provided by Gibco (Gaithersburg, USA). MC3T3-E1 cells were cultured with α-MEM supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere.
High-glucose (25.0 mmol/L) medium was produced by adding extra D-glucose purchased from Sigma–Aldrich Co., Ltd. (Shanghai, China) to the ordinary medium. β-glycerophosphate and ascorbic acid were purchased from Aladdin Co., Ltd. (Shanghai, China), and β-glycerophosphate (10 mM) and ascorbic acid (50 mg/mL) dissolved in α-MEM supplemented with 10% FBS acted as the osteogenesis medium. Anti-GSK3β, anti-p-GSK3β (S9), anti-β-catenin, anti-Cyclin D1, anti-Cmyc, and anti-Runx2 were purchased from Abcam (Cambridge, UK). Alizarin Red S staining solution, propidium iodide solution and DAPI solution were purchased from Solarbio Co., Ltd. (Beijing, China). Furthermore, Beyotime Biotechnology (Suzhou, China) provided an EdU Cell Proliferation Kit with Alexa Fluor 555, BCIP/NBT Alkaline Phosphatase Colour Development Kit, Alkaline Phosphatase Assay Kit, anti-β-actin, HRP-labelled secondary antibody and Enhanced Chemiluminescence Kit. XAV939 was purchased from MedChemExpress Company (New Jersey, USA).

The cells were either treated by ELAE at an optimum concentration based on the result of the cell proliferation assay or treated with a plain culture medium (Thermo, USA) and incubated for 24 h. All culture media were removed, and cells were washed with phosphate-buffered saline buffer (Thermo, USA). The cells were fixed by incubating with 4% paraformaldehyde for 15 min at room temperature. After washing with Millipore purified water three times, the fixed cells were stained with Alizarin red S staining solution (Solarbio, China) for 30 min at room temperature and washed with purified water three times. Images were taken with an optical microscope (Olympus, Japan) accompanied by a digital camera. Quantitative analysis was carried out after dissolving the stained cells with cetylpyridinium chloride monohydrate reagent (Solarbio, China) for 1 h at room temperature, using a plate reader (Life science, USA) at an optical density (OD) of 562 nm.

BMSCs were subjected to induction for osteoblastogenesis for 20 days. Before staining, cells were rinsed and fixed, and 1% alizarin red S staining solution (pH 4.2, Solarbio, Beijing, China) was used for staining. An inverted microscope was used to observe calcification nodules and capture images (Leica, Weztlar, Germany, magnification 100×).