MC3T3-E1 cells were seeded in 6-well plates and cultured in osteogenic induction medium with stimulation for 14 days. Alkaline phosphatase (ALP) staining was performed with a BCIP/NBT (nitro-blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate) alkaline phosphatase colour development kit (C3206, Beyotime, China) according to the manufacturer’s instructions. When the cells were induced for 28 days, Alizarin Red S (ARS) staining was performed to detect calcium deposits with modified Alizarin Red S stain kit for calcium (G3280, Solarbio, China). Stained plates were photographed using a digital camera. Images of stained cells were captured under a light microscope (BX41, Olympus, Japan) and five randomly selected fields (× 10 magnification) were photographed in each well, analysed by Image J software (version 1.53n, National Institutes of Health, USA) according to previous protocol [27 (link)–29 (link)].
Alizarin red s stain kit for calcium
Manufactured by Solarbio
Sourced in China
The Alizarin Red S stain kit is a laboratory reagent used for the detection and visualization of calcium in various samples. It provides a simple and effective method for staining calcium-containing structures, which can be useful in a range of applications, such as histology, cell biology, and biochemistry.
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2 protocols using alizarin red s stain kit for calcium
1
Osteoblast Differentiation Assays
2
Osteogenic differentiation of MC3T3-E1 cells
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MC3T3-E1 cells were seeded in six-well plates (5 × 103 cells/cm2) and cultured in osteogenic induction medium with stimulation for 14 days. To induce osteogenic differentiation, 50 μg/ml L-ascorbic acid (MilliporeSigma, USA), 10 mM β-glycerophosphate (MilliporeSigma), and 10 nM dexamethasone (MilliporeSigma) were added in α-MEM. Alkaline phosphatase (ALP) staining was performed with a nitro-blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (BCIP/NBT) ALP colour development kit (C3206; Beyotime, China) according to the manufacturer’s instructions. Alizarin Red S staining was performed to detect calcium deposits using a modified Alizarin Red S stain kit for calcium (G3280; Solarbio, China). Stained plates were photographed using a digital camera. Images of stained cells were captured under a light microscope (BX41; Olympus), and five randomly selected fields (× 10 magnification) were photographed in each well for analysis using ImageJ software (version 1.53 n; National Institutes of Health, USA).
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