Figure 5: CIE promotes ascorbic acid and β-glycerol phosphate mediated osteoblast differentiation. (a) Primary osteoblasts were treated with various concentrations of CIE for 7 days in the presence of 50 μg/mL ascorbic acid and 10 mM β-glycerol phosphate. ALP-positive cells were stained with ALP solution. (b) Primary osteoblasts were treated with various concentrations of CIE for 21 days. Calcium accumulation within the osteoblasts was stained with ARS solution. Stained calcium deposits were dissolved by 10% CPC buffer to measure the level of staining. (c) Primary osteoblasts were stimulated with ascorbic acid (50 μg/mL) and β-glycerol phosphate in the presence of CIE (50 μg/mL) or DMSO. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of Runx2, ALP, Col1α, and OPN were evaluated by real-time PCR. , , and .