2.3. Alkaline Phosphatase (ALP) Activity Assay and ALP Staining

Levels of early osteoblast differentiation were determined by alkaline phosphatase (ALP) activity assay and ALP staining analysis. For ALP activity assay, cells were cultured in 24-well plates and induced for early osteoblast differentiation. Cell extracts were prepared with CytoBuster™ protein extraction reagent and centrifuged at 15,000g for 5 minutes. Then, 10 μL cell extracts were added and incubated with 100 μL of p-nitrophenyl phosphate (p-NPP) for 30 min and stopped by adding 100 μL of 1 N NaOH, and then, absorbance was read at 405 nm. The protein content of cell extracts was measured by the BCA method. The OD405 values was standardized by protein content, and then, the relative ALP activity was presented as fold change to the control group. For ALP staining, cells were washed with PBS twice, fixed with 70% ethanol for 20 min. After washing three times with PBS, cells were incubated with a mixture of 0.1 mg/mL napthol AS-MX phosphate and 0.6 mg/mL Fast Blue BB salt for 30 min at room temperature.