Microplate reader

Alkaline phosphatase (ALP) is major marker of the early and middle stage of osteoblast differentiation (Lee et al., 2017 (link)). To visualize ALP abundance, after 14 days of treatment, cells were washed with PBS and fixed in 10% formalin for 10 min. The fixed cells were incubated in a solution containing 1 mg/ml Fast Blue RR salt and 0.4 mg/ml naphthol AS‐MX phosphate disodium salt (Thermo Fisher Scientific, Waltham, MA, USA) at pH 8.4 for 15 min. The reactions were stopped by rinsing with deionized water, and cells were photographed under a light microscope (Motic Microscopes, Xiamen, China). To measure ALP activity, an enzymatic ALP activity assay (Abcam, Cambridge, UK) was used according to the instructions provided by the manufacturer. Briefly, cells were harvested with a cell lysis solution, a p‐nitrophenyl phosphate (pNPP) phosphatase substrate was applied to the samples, and absorbance was read at 405 nm using a microplate reader (Norgen BioTek). Absorbance data were corrected to background and compared with control.

To measure calcium deposition, after 21 days of treatment, cells were washed with Dulbecco's phosphate‐buffered saline (DPBS), then fixed with 10% formalin and stained with 2% Alizarin Red S (ARS) solution (EMD) for 15 min. The ARS solution was removed, and the cells were then washed five times with water. Representative images were taken using phase‐contrast microscopy (Motic Microscopes). The dye was extracted using 10% acetic acid, cells were separated, and supernatant was transferred to a 96‐well plate and absorbance was read at 405 nm in a microplate reader (Norgen BioTek). Absorbance data were corrected for background and compared with control.