Figure 6.

Effect of well size on alizarin red S staining of human SaOs-2 osteoblasts. Human osteoblasts were stimulated with osteoblast differentiation medium (ODM+) and in combination with calcium chloride (5 mM) in 24- or 96-well plates for three weeks before alizarin red S staining was performed. ODM− served as a negative control. (A) Representative pictures of alizarin red S-stained SaOs-2 osteoblasts assessed with transmitted light microscopy at 20x magnification are shown. Scale bar is 50 µm. (B) Concentrations of alizarin red S were calculated photometrically and are represented relative to the ODM+ reference of the 96-well plate. Means ± standard deviation from three independent experiments using sextuplicates are shown. * Indicates a significant difference (p < 0.001).