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Alizarin red staining and quantification

YG Yingnan Geng
HD Huichuan Duan
LX Liang Xu
NW Nevin Witman
BY Bingqian Yan
ZY Zheyuan Yu
HW Huijing Wang
YT Yao Tan
LL Liqin Lin
DL Dong Li
SB Shanshan Bai
RF Regina Fritsche-Danielson
JY Jie Yuan
KC Kenneth Chien
MW Min Wei
WF Wei Fu
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To detect mineralization, BMSCs were subject to Alizarin red staining at 14 dpoi, as described previously57. After removing the medium, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. Then cells were rinsed with PBS twice, and stained with 0.2% Alizarin Red S solution (pH 4.2) for 5 min, followed by three times PBS wash extensively to remove unspecific staining or verisimilar sediments. Calcified nodules are stained as red spots and were photographed by a microscope. To quantify the calcium deposition, after washing with dH2O, the stain was solubilized with 10% cetylpyridinium chloride monohydrate (CPC, Sigma-Aldrich) in 0.1 M PBS (pH 7.0) by shaking for 15 min and the absorbance was read at 570 nm58. The calcium deposition is expressed as optical density (OD).

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