Optimization of the Alizarin Red S Assay by Enhancing Mineralization of Osteoblasts

Abstract

The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are several restrictions to this method, e.g., moderate sensitivity makes it difficult to uncover slight but significant effects of potentially clinically relevant substances. Therefore, an adaptation of the staining method is appropriate and might be obtained by increasing the mineralization ability of osteoblasts. In this study, cell culture experiments with human (SaOs-2) and murine (MC3T3-E1) osteoblasts were performed under the addition of increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM). After three or four weeks, the mineralization matrix was stained with alizarin red S and the concentration was quantified photometrically. Only calcium chloride was able to significantly increase mineralization, and therefore enhanced the sensitivity of the alizarin red S staining in a dose-dependent manner in both osteoblastic cell lines as well as independent of the cell culture well surface area. This cost- and time-efficient optimization enables a more sensitive analysis of potentially clinically relevant substances in future bone research.

Keywords: alizarin red S assay; bone; calcitonin; calcium chloride; mineralization; osteoblasts.

Figure 1

Alizarin red S staining of murine osteoblasts (MC3T3-E1) after incubation with calcium chloride or calcitonin. Cells were incubated with increasing calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM) concentrations for three (left panel) or four weeks (right panel) and subsequently stained with alizarin red S. Each condition was assessed in triplicate in three independent assays (n = 9). ODM− served as negative control (n = 6), ODM+ (n = 12) as reference, and acetic acid (n = 6) was used as an additional control as calcitonin was dissolved in acetic acid. Representative pictures of transmitted light microscopy at 20× magnification are shown. Scale bar is 50 µm. ODM; osteoblast differentiation medium.

Figure 2

Photometric quantification of alizarin red S in murine MC3T3-E1 cells. Cells were incubated with increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM) for three or four weeks. Concentrations of alizarin red S are represented relative to the ODM+ reference after three weeks. Values represent means of three independent experiments in triplicate (n = 9) except for ODM+ reference (n = 12), the negative control (ODM−; n = 6) and the acetic acid control (n = 6) ± standard deviation. ODM; osteoblast differentiation medium. * Indicates a significant difference (p < 0.05).

Figure 3

Alizarin red S staining of human osteoblasts (SaOs-2) after incubation with calcium chloride or calcitonin. Cells were incubated with increasing calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM) concentrations for three (left panel) and four weeks (right panel), and subsequently stained with alizarin red S. Each condition was assessed in triplicate in three independent experiments (n = 9). ODM− served as a negative control (n = 6), ODM+ (n = 12) as reference, and acetic acid (n = 6) was used as an additional control as calcitonin was dissolved in acetic acid. Representative pictures of transmitted light microscopy at 20x magnification are shown. Scale bar is 50 µm. ODM; osteoblast differentiation medium.

Figure 4

Photometric quantification of alizarin red S concentrations in human SaOs-2 cells. Cells were incubated with increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM) for three or four weeks. Concentrations of alizarin red S are represented relative to the ODM+ reference after three weeks. Values represent means of three independent assays in triplicate (n = 9) except for the ODM+ reference (n = 12), the negative control (ODM−; n = 6) and the acetic acid control (n = 6) ± standard deviation. ODM; osteoblast differentiation medium. * Indicates a significant difference (p < 0.05).

Figure 5

Effect of well size on alizarin red S staining of murine MC3T3-E1 osteoblasts. Murine 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 MC3T3-E1 osteoblasts assessed with transmitted light microscopy at 20× 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).

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).