2.4. Alkaline Phosphatase (ALP) Staining

The culture medium was removed after osteogenic differentiation; alkaline phosphatase (ALP) staining was performed to identify MSCs according to the instructions of the ALP staining kit (Sigma, USA). The cells were washed twice with phosphate-buffered saline (PBS) and fixed at room temperature with 4% paraformaldehyde for 10 minutes. After fixation, the cells were rinsed again with PBS. An appropriate amount of ALP dye solution was added to the fixed cells in a 6-well plate in the dark and incubated at room temperature for approximately 15-30 minutes. The staining was monitored under a fluorescence microscope every 5 minutes. After the staining was stable, the plate was rinsed with PBS again for final observation. Osteogenic differentiation was confirmed by deepening cytoplasmic staining.