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Alizarin Red S staining and quantification
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Calcium deposition, at day 12 of osteoblastic differentiation, was measured using Alizarin Red staining. Osteoblasts were fixed with 70% ice-cold ethanol for 1 h at -20 °C before addition of AR-S (40 mM; Sigma-Aldrich) dissolved in distilled water, pH 4.17. The cells were stained for 10 min at room temperature. The level of calcium deposition was quantified by elution of AR-S following incubation in 10% cetylpyridinium chloride (Sigma-Aldrich) for 1 h at room temperature. The absorbance of the eluted dye was assessed at 570 nm in a FLU Ostar Omega plate reader.
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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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