Cetylpyridinium chloride

After inducing osteogenic differentiation for 14 days, the cells were collected and fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 30 min and washed thrice for 1 min each. Incubate the cells with 2% pH 4.2 Alizarin Red staining solution (Solarbio, Beijing, China) at room temperature in dark for 20 min. Staining was observed under an inverted microscope (Leica DMIRB). To quantify the degree of mineralization of BMSCs, the stain was solubilized with cetylpyridinium chloride (Solarbio, Beijing, China) and quantified using a spectrophotometer at 570 nm.
²⁶ (link) ARS staining and quantitative experiments were repeated 3 times for each section; a total of 39 images were captured throughout the entire experiment.

For the osteogenic differentiation assay, PDLSCs were cultured in osteogenic medium containing α-MEM (BI), 10% FBS (BI), 10 nM dexamethasone (Solarbio), 10 mM β-glycerophosphate (Solarbio) and 50 mg/L ascorbic acid (Solarbio). After 4 weeks, the cells were fixed with paraformaldehyde and stained with Alizarin Red solution (Sigma, St. Louis, MO, USA) to detect mineralized nodules. To analyse the intensity of Alizarin red staining, mineralized nodules were dissolved in 10% cetylpyridinium chloride (Solarbio) and quantified using a microplate reader at 562 nm. When necessary, Ly294002 (#9901, CST) (an inhibitor of Akt phosphorylation) or SC79 (HY-18749, MCE, USA) (a promoter of Akt phosphorylation) was added to the osteogenic medium at concentration of 10 µmol/L or 5 µg/mL, respectively.
For the adipogenic differentiation assay, PDLSCs were cultured in adipogenic medium containing α-MEM (BI), 10% FBS (BI), 1 µM dexamethasone (Solarbio), 0.2 mM indomethacin (Solarbio), 0.01 g/L insulin (Solarbio) and 0.5 mM isobutyl-methylxanthine (Solarbio). Four weeks later, the cells were fixed with paraformaldehyde and stained with oil red O (Solarbio) to detect lipid droplets.