To determine the initial osteogenic expression of ALP, 1 × 105 hMSCs were seeded in 12-well plates. After 24 h, the medium was changed to osteogenic medium for the extraction of control and experimental groups, which was replaced every 2–3 days. After culturing for 14 days, ALP staining (Sigma Aldrich) was performed according to the manufacturer’s instructions. In addition, ALP activity was determine using a SensoLyte pNPP alkaline phosphatase assay kit according to the manufacturer’s instructions. The total protein was normalized using a PierceTM BCA Protein Assay kit according to the manufacturer’s instruction.
To perform the mineralization after culturing for 28 days, Alizarin Red S staining (ARS) was performed in previous study [32 (link)]. The cells were fixed with 70% ice-cold alcohol for 1 h. The fixed cells were stained for 15 min with 2% ARS solution, pH 4.2 adjusted to ammonium hydroxide solution at 25 °C. After washing three times with distilled water, 10% cetylpyridinium chloride solution was added, and the absorbance of the solution was measured at 560 nm. The tests were performed three times.
To perform the mineralization after culturing for 28 days, Alizarin Red S staining (ARS) was performed in previous study [32 (link)]. The cells were fixed with 70% ice-cold alcohol for 1 h. The fixed cells were stained for 15 min with 2% ARS solution, pH 4.2 adjusted to ammonium hydroxide solution at 25 °C. After washing three times with distilled water, 10% cetylpyridinium chloride solution was added, and the absorbance of the solution was measured at 560 nm. The tests were performed three times.
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