4.8. ALP Staining Assay

Osteoblast differentiation was induced using OS containing 50 μg/mL L-AA and 10 mM β-GP with Obac (1 and 10 μM) for 7 days. ALP staining assay was performed as previously described [47]. Cells were fixed in 10% formalin, rinsed with distilled water, and incubated at 37 °C for 1 h in substrate solution for ALP reaction (Takara Bio Inc., Japan). The level of ALP staining was observed using a scanner and colorimetric detector (ProteinSimple Inc., Santa Clara, CA, USA).