Introduction

Alizarin red S (Figure 1) is a sulfonated, water-soluble derivative of alizarin. Alizarin red S staining has been used for decades to evaluate calcium-rich deposits by cells in culture. It is particularly versatile in that the dye can be extracted from the stained monolayer and assayed. In addition，Alizarin red S is considered the gold standard for the detection and quantification of mineralization since this anthraquinone derivative reacts more specifically with calcium cations through a chelation process. An alizarin red S-calcium complex is formed thereby, resulting in a bright red stain that can also be observed visually. This method consists of the fixation of the cells with formalin, followed by staining with alizarin red S dye and examining the staining under a phase-contrast microscope. For quantitative analysis, the dye can be extracted and quantified by a colorimetric assay. This article summarizes its mainly recent research.

1. An Alizarin red-based assay of mineralization by adherent cells inculture: comparison with cetylpyridinium chloride extraction

This study describes a sensitive method for the recovery and semiquantification of Alizarin red S  in a stained monolayer by acetic acid extraction and neutralization with ammonium hydroxide followed by colorimetric detection at 405 nm. This method was three times more sensitive than an older method involving cetylpyridinium chloride (CPC) extraction and resulted in a better signal to noise ratio, especially for weakly stained monolayers. The assay facilitates detailed inspection of mineralization by phase microscopy and semiquantiWcation of the entire monolayer by extractionand quantification. The sensitivity of the assay is improved by the extraction of the calcified mineral at low pH and, since the mineralis already stained in a quantitative manner, there is no requirement for an additional colorimetric quantification step. Furthermore,the linear range is much wider than those of conventional assays for calcium, making dilutions of mineral extracts prior to measurement unnecessary. It has a wide range of potential uses including tumor characterization, mesenchymal stem cell evaluation, and osteogenic compound screening. Although more labor intensive than CPC extraction, the protocol is more sensitive and yields more reliable results for weakly mineralizing samples.[1]

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

The alizarin red S assay is considered the gold standard for quanti?cation of osteoblast mineralization and is thus widely used among scientists. However, there are several restrictions to this method, e.g., moderate sensitivity makes it dif?cult to uncover slight but signi?cant 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 quanti?ed 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.[2]

3. Excited State Dynamics of Alizarin Red S Nanoparticles in Solution

This work presents femtosecond studies of alizarin red S (ARS) nanoparticles in comparison to ARS in aqueous solution and to alizarin in DMSO. The femtosecond studies cover a probing spectral range of 350–750nm using different excitation wavelengths, taking into account the variation of the absorption spectra with the pH values of the solvent. Stationary absorption spectra show slight differences between solution and nanoparticles. Excitation at 530nm results in low and noisy responses, therefore, we additionally recorded transient spectra of the nanoparticles at λex = 267nm. While the results in DMSO are comparable to previous studies in non-aqueous solvents, we report a relatively fast relaxation of 14ps in [La(OH)2][ARS] nanoparticles in aqueous solution after excitation at 530 nm, which is similar to Na(ARS) solution (19ps). The dynamics changed with lower pH, but still without signi?cant differences between nanoparticles and solution. We propose [La(OH)2][ARS] nanoparticles as a suitable alternative to dissolved molecules with similar spectroscopic properties, for example, with regard to biomarker applications.[3]

4. Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method

The researchers identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.[4]

References

[1] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem. 2004;329(1):77-84. doi:10.1016/j.ab.2004.02.002

[2] Bernar A, Gebetsberger JV, Bauer M, Streif W, Schirmer M. Optimization of the Alizarin Red S Assay by Enhancing Mineralization of Osteoblasts. Int J Mol Sci. 2022;24(1):723. Published 2022 Dec 31. doi:10.3390/ijms24010723

[3] Rauthe P, Sabljo K, Vogelbacher MK, Feldmann C, Unterreiner AN. Excited State Dynamics of Alizarin Red S Nanoparticles in Solution. Molecules. 2023;28(15):5633. Published 2023 Jul 25. doi:10.3390/molecules28155633

[4] Fischbach J, Loh Q, Bier FF, Lim TS, Frohme M, Gl?kler J. Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method. Sci Rep. 2017;7:45085. Published 2017 Mar 24. doi:10.1038/srep45085