hBMSCs were subjected to osteogenic induction for 14 days, washed twice with PBS, and fixed with 4% PFA for 15 min. Next, they were washed with diH2O and stained with 40 mM ARS (ScienCell, US) at 37°C for 15 min. After dyeing, the cells were washed with diH2O, and images were obtained under the microscope (Leica, DMIRB, Germany). The stained cells were added with 10% acetic acid and 10% ammonium hydroxide from an Alizarin Red S staining quantification assay kit (ScienCell, US). The absorbance at 405 nm was determined and used to analyze the ARS concentration.
Alizarin red s staining quantification assay kit
Manufactured by ScienCell
Sourced in United States
The Alizarin Red S Staining Quantification Assay Kit is a laboratory tool used to quantify the amount of calcified extracellular matrix or mineralized deposits in cell cultures. The kit provides a colorimetric method to measure the intensity of the Alizarin Red S stain, which binds to calcium deposits in the samples.
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Lab products found in correlation
Dm irb, by Leica camera (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Inverted microscope, by Leica camera (1 mentions)
Alizarin red solution, by Merck Group (1 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (1 mentions)
Osteoimage mineralization assay kit, by Lonza (1 mentions)
Mc3t3 e1, by Merck Group (1 mentions)
β glycerophosphate disodium salt hydrate, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Alizarin red s staining kit, by ScienCell (1 mentions)
β glycerophosphate, by Thermo Fisher Scientific (1 mentions)
Alkaline phosphatase assay kit, by Abcam (1 mentions)
Infinite m200, by Tecan (1 mentions)
5 protocols using alizarin red s staining quantification assay kit
1
Osteogenic Differentiation of hBMSCs
2
Alizarin Red Staining for Osteogenic Differentiation
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After 15 days of osteogenic stimulation, JPCs were fixed with fixation buffer (Biolegend, California, USA) and stained with 40 mM Alizarin red solution (pH 4.2, Sigma-Aldrich) for 20 min. Unbound dye was removed by washing with deionized water and images were taken using an inverted microscope (Leica, Wetzlar, Germany). The stained plates were quantified using alizarin red S staining quantification assay kit (ScienCell, California, USA) following the manufacturer’s instructions and photometrical quantification of the alizarin staining was performed at a wavelength of 405 nm using a microplate reader (Biotek, Bad Friedrichshall, Germany). JPCs were further stained for hydroxyapatite detection using the OsteoImage mineralization assay kit (Lonza, Basel, Switzerland) following the manufacturer’s instructions. Images were taken using an Axio Observer Z1 fluorescence microscope (Zeiss, Oberkochen, Germany).
3
Osteoblastic Differentiation of MC3T3-E1 Cells
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The osteoblastic cell line, MC3T3-E1, was purchased from BCRC (Hsinchu, Taiwan) and was grown in αMEM supplemented with 10% FBS without ribonucleosides and deoxyribonucleosides at 37 °C. For osteogenic differentiation, MC3T3-E1 cells were cultured in growth medium supplemented with L-ascorbic acid (50 µg/mL; Sigma-Aldrich) and β–glycerophosphate disodium salt hydrate (10 mM; Sigma-Aldrich) for 20 days. The differentiation medium was refreshed every 3–4 days. After differentiation, the calcium deposits were stained and quantified using the Alizarin Red S Staining Quantification Assay Kit (ScienCell Research Laboratories, Carlsbad, CA, USA).
4
Osteogenic Differentiation Assay Protocol
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Before the analysis of osteogenic properties, cells were treated with osteogenic induction medium consisting of DMEM with 10% FBS, 25 mg/ml ascorbate-2 phosphate, 10−8 M dexamethasone, and 5 mM β-glycerophosphate (All from Gibco, USA) for the indicated time (otherwise 2 weeks). After induction, cells were fixed with 4% paraformaldehyde, after few washes with PBS, cells were stained according to the manufacturer’s instruction. We used Alizarin Red S Staining Kit (#0223, Sciencell, San Diego, USA) for calcium deposition analysis, Alizarin Red S Staining Quantification Assay kit (#8678, Sciencell, San Diego, USA) was used for quantification. Alkaline phosphatase staining kit (1102-100, Sidansai, Shanghai, China) was used for cellular ALP staining, and Alkaline Phosphatase Assay kit (ab83369, Abcam, Beverly, USA) for quantification. The average value of OD405nm of each sample were calculated, and the average value of OD570nm of each sample were also calculated as background control. Values of OD405nm were divided by OD570nm to generate a standardized OD value for each sample for further comparison. Infinite™ M200 (Tecan, USA) microplate plate reader was used for absorbance quantification, and three replicates were done to generate the average value.
5
Quantifying Alizarin Red S Staining in WJ-MSC Osteogenesis
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WJ-MSCs were seeded at 5×104 cells/well in a 6-well plate. The culture media was changed as indicated by the Stempro Osteocyte Differentiation Kit (Gibco) and WJ-MSCs were cultured for 23 days in differentiation media with or without NA. The cultured cells were fixed with 4% PFA, and the differentiated cells were using Alizarin Red S Staining Quantification Assay Kit (ScienCell), according to the manufacturer’s instructions. Alizarin Red S was dissolved in 10% acetic acid, and optical density was measured at 405 nm using Multiskan SkyHigh.
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