For ALP staining, after induction for 14 days, cells were fixed with 95% ethanol and stained with BCIP/NBT solution according to the manufacturer's protocol (Beyotime Institute of Biotechnology) at room temperature for 2 h. The ALP-positive cells were stained blue/purple. Stained cells were visualized using the Canon IXUS210 camera (Canon, Inc.; magnification). In addition, an Alkaline Phosphatase Staining Kit (BestBio, Inc.) was used to detect the ALP activity, according to the manufacturer's protocol.
For alizarin red staining, after induction for 14 days, cells were washed one or two times with phosphate-buffered saline (PBS), fixed with 95% ethanol for 10 min, washed one or two times with PBS again, covered, and stained with 0.1% alizarin red solution for 10 min. Finally, they were rinsed with PBS and observed under an inverted light microscope. For quantification analysis, 10% hexadecyl pyridinium chloride monohydrate (CPC; Sigma-Aldrich; Merck KGaA) was used to dissolve the mineralized nodules and then the absorbance was measured at 540 nm using a Multiskan™ FC spectrophotometer (Thermo Fisher Scientific, Inc.).
For alizarin red staining, after induction for 14 days, cells were washed one or two times with phosphate-buffered saline (PBS), fixed with 95% ethanol for 10 min, washed one or two times with PBS again, covered, and stained with 0.1% alizarin red solution for 10 min. Finally, they were rinsed with PBS and observed under an inverted light microscope. For quantification analysis, 10% hexadecyl pyridinium chloride monohydrate (CPC; Sigma-Aldrich; Merck KGaA) was used to dissolve the mineralized nodules and then the absorbance was measured at 540 nm using a Multiskan™ FC spectrophotometer (Thermo Fisher Scientific, Inc.).