Alkaline phosphatase alp assay kit

Chondroitin 4-sulfate sodium salt (CS, cell culture, ≥ 85%), gelatin (type B, gel strength ~100 g Bloom), sodium tetraborate decahydrate (Na₂B₄O₇·10H₂O, GR, 99.5%), ammonia solution (AR, 25–28%), borax, sodium periodate (AR, ≥99.5%) and calcein (AM) were purchased from Aladdin (Shanghai, China). Tetraethyl orthosilicate (TEOS, AR, 98%) was purchased from Sigma-Aldrich (St. Louis, USA). Hexadecyl trimethyl ammonium bromide (CTAB, AR, ≥99%) was purchased from Shanghai Sinopharm Chemical Reagent Co. Ltd. Calcium nitrate (CN, AR, 99%) was provided by Tianjin Fuchen Chemical Reagent Factory. Ethyl acetate (EA, AR, ≥99.5%) and absolute ethanol (AR, ≥99.7%) were purchased from Guangdong Guanghua Technology Co. Ltd. Rat bone marrow mesenchymal stem cells (BMSCs) were provided by the Stem Cell Bank, Chinese Academy of Sciences. Dulbecco's modified Eagle medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from HyClone, USA. Actin-Tracker Green, 4′, 6-diamidino-2-phenylindole (DAPI), Propidium Iodide (PI), Cell Counting Kit-8 (CCK-8) and Alkaline Phosphatase Assay Kit (ALP) were purchased from Beyotime Biotechnology.

Osteogenic induction was performed by culturing cells in an osteogenic differentiation medium (Cyagen Biosciences) containing 10% (v/v) FBS, 1% (v/v) penicillin–streptomycin, 2 mM l-glutamine, 50 μM ascorbate, 10 mM β-glycerophosphate, and 100 nM dexamethasone. The culture medium was changed every 2–3 days. The BMSCs were induced in the osteogenic differentiation medium for 14 days. Cells were washed thrice with PBS, and then fixed with 4% PFA for 15 min. Finally, cells were stained with an Alkaline Phosphatase Assay Kit (Alp; Beyotime, Shanghai, China, #C3206), according to the manufacturer’s instructions.
Alizarin Red S staining was performed after 21 days of induction. After fixing with 4% PFA, each well was treated with Alizarin Red S solution (Cyagen Biosciences) and incubated in the dark for 30 min. Then, the images were obtained by microscopy. Finally, quantification of calcium deposition was performed by elution of AR-S with 10% (W/V) cetylpyridinium chloride in 10 mM sodium phosphate (PH 7.0) for 1 h at room temperature, and absorbance of the eluted dye was measured at 570 nm.

As shown in Table 1, well‐defined ssDNAs were synthesized by TAKARA (Dalian, China). LPS and alkaline phosphatase (ALP) assay kit, which were used to induce the inflammatory environment, were obtained from Beyotime (Shanghai, China). Osteogenic differentiation medium was purchased from Cyagen (Shanghai, China). Primary antibodies and secondary antibodies were purchased separately from Abcam (Cambridge, UK) and Signalway Antibody (Shanghai, China).