2.4. Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) Staining

For ALP staining, after formalin fixation (10%) and rinsing twice with PBS (pH 7.4), osteoblasts were maintained with ALP substrate solution, naphthol AS-MX (0.1 mg/ml, Sigma-Aldrich, USA), and fast violet B salt (0.6 mg/ml, Sigma-Aldrich, USA) in 0.1 M Tris-HCl (pH 8.5). In addition, ALP activity was assayed by a LabAssay ALP kit (Wako, Japan) after osteoblasts being rinsed twice with PBS and lysed in Mammalian Protein Extraction Reagent (Pierce, USA) per the specification. Besides, protein quantification using the BCA protein assay kit (Pierce, USA) was implemented under the manufacturer's protocol.

For ARS staining, cells were rinsed twice with PBS and then fixed for 10 min in 2% formaldehyde, followed by 30 min of cell staining with 0.2% ARS solution at 37°C. Each well was rinsed 3 times with distilled water and pictured. The ARS red staining indicated calcium deposition.