After culturing in osteogenic media for 7, 14, and 21 days, MOVAS cells were fixed in 4% paraformaldehyde and stained with 2% Alizarin red S (ARS) solution (pH 4.2, ScienCell, Carlsbad, CA, USA) for 30 minutes at room temperature, following the manufacturer’s protocol. Brightfield images were taken of calcium-stained cells using a Leica DMi8 (Leica, Wetzlar, Germany). Cellular calcification was quantified via ARS dye extraction by incubating the cells with 10% acetic acid for 30 minutes with constant agitation.30 (link) Cells and calcium deposits were then collected, heated to 85°C for 10 minutes, and centrifuged at 20,000 x g for 15 minutes. The supernatant containing the extracted ARS dye was collected and absorbance was measured at 405 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA, N=6).
Alizarin red s solution
Manufactured by ScienCell
Sourced in United States
Alizarin Red S solution is a chemical reagent commonly used in laboratory settings. It serves as a staining agent for the detection and visualization of calcium deposits in biological samples. The solution provides a distinct red color when it interacts with calcium, allowing for easy identification and analysis of calcium-rich structures.
Automatically generated - may contain errors
Lab products found in correlation
Ckx41sf, by Olympus (2 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Spectramax m5 microplate reader, by Molecular Devices (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
K412 500, by Abcam (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Spectramax 190 plate reader, by Molecular Devices (1 mentions)
Fast violet b salt, by Merck Group (1 mentions)
Tracp alp assay kit, by Takara Bio (1 mentions)
Calcium liquicolor assay, by EKF Diagnostics (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Wst 1 reagent, by Roche (1 mentions)
8 protocols using alizarin red s solution
1
Quantifying Calcium Deposition in Osteogenic MOVAS Cells
2
Calcification Induction in Vascular Cells
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For calcification induction, A10 and primary VSMCs (passages 3–5) were seeded on a coverslip and grown in GM until they reached 80% confluence. They were then transferred to OM consisting of DMEM containing CaCl2 (2.85 mmol/L), β-glycerophosphate disodium salt hydrate (5 mmol/L), 10% fetal bovine serum, and penicillin–streptomycin for 7 days. Media were replaced every other day. After the medium was removed, the cells were washed using PBS, fixed with 10% formalin, and stained with alizarin red S (ARS) solution (ScienCell Research Laboratories, Carlsbad, CA, USA) for 30 min. After images were captured, calcified minerals were extracted with 10% acetic acid and neutralized using 10% ammonium hydroxide. ARS standards, ranging from 30 µM to 4 mM, were prepared. Absorbance of the sample and standards was measured at 405 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Based on the calibrated optical density (OD)405nm value of the ARS standard, a standard curve was plotted as a function of ARS concentration. The ARS concentrations in the samples were calculated according to the equation of the trend line.
3
Osteogenic Differentiation of Stem Cells
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To access the osteogenic differentiation of stem cells, absorbance at 405 nm was measured using an alkaline phosphatase assay kit (K412-500, BioVision, Inc., Milpitas, CA, USA) according to the manufacturer’s protocol after 1, 3, 7 and 14 days of cell culture using a microplate reader (BioTek Instruments Inc.).
The cells were washed, fixed and colored with 2% Alizarin Red S Solution (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) after 7 and 14 days of cell culture. The stained cells were visualized using a microscope (CKX41SF, Olympus Corporation). Ten percent cetylpyridinium chloride (Sigma-Aldrich Co.) was used to dissolve the bound dye and quantification was performed spectrophotometrically at 560 nm.
The cells were washed, fixed and colored with 2% Alizarin Red S Solution (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) after 7 and 14 days of cell culture. The stained cells were visualized using a microscope (CKX41SF, Olympus Corporation). Ten percent cetylpyridinium chloride (Sigma-Aldrich Co.) was used to dissolve the bound dye and quantification was performed spectrophotometrically at 560 nm.
4
Quantitative Alizarin Red S Assay
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Cells were washed twice with phosphate‐buffered saline and fixed with 4% paraformaldehyde at 25°C for 15 minutes. The cells were then washed three times with distilled water, 2% Alizarin Red S solution (ScienCell) was added to each well, and the cells were incubated at 25°C for 30 minutes. The cells were then washed five times with distilled water and images were captured using a Canon PowerShot SD990 IS camera. After imaging, cells were stored at −20°C until assay. Cells were treated with 10% acetic acid, scraped from the plate, and heated to 85°C. After centrifugation, the supernatant was neutralized with 10% ammonium hydroxide, and absorbance was read at 405 nm on a SpectraMax 190 plate reader (Molecular Devices).
5
Alizarin Red S Staining Protocol
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Alizarin Red S staining was conducted on Days 7 and 14. Washing, fixing, and staining with 2% Alizarin Red S Solution (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) were all performed on the cells and scrutinized under a microscope (CKX41SF, Olympus Corporation) [19 (link)]. Using image analysis software to measure the staining’s intensity, the relative values of Alizarin Red S staining were calculated (ImageJ, National Institutes of Health, Bethesda, MD, USA) [20 (link)].
6
Quantitative Alizarin Red S Staining
7
Osteoblast Differentiation and Mineralization
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Cells were seeded onto a 12-well plate, with 1 × 104 MC3T3-E1 cells per well. OGFs (100 nM dexamethasone, 0.05 mM ascorbic acid 2-phosphate, and 10 mM b-glycerophosphate; Sigma-Aldrich, St. Louis, MO, USA) were added to trigger osteoblast differentiation under different treatments. After the cells had been treated, they were fixed in 60% citrate-buffered acetone and then incubated with a mixture of Fast Violet B Salt and naphthol AS-MX phosphate alkaline solutions (Sigma-Aldrich, St. Louis, MO, USA) for the detection of ALP expression. ALP activity and the total number of cells were measured using the TRACP & ALP assay kit (TakaRa Bio, Kusatasu, Shiga, Japan) and water-soluble tetrazolium salt (WST-1) reagent (Roche, Basel, Switzerland), respectively, in accordance with the manufacturers’ instructions. Cells were fixed with 10% paraformaldehyde after treatment, and then a 2% Alizarin Red S solution (ScienCell, Carlsbad, CA, USA) was used for detecting calcification. Calcium LiquiColor Assay (Stanbio Laboratory, Boeme, TX, USA) and WST-1 reagent (Roche, Basel, Switzerland) were respectively used in accordance with the manufacturers’ instructions to determine the calcium content and number of cells after treatment.
8
Quantification of Alizarin Red Staining
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Cells were fixed with 1 mL 10% Formalin for 10 min at RT. Fixed cells were then washed twice with PBS before staining with 1 mL of 2% Alizarin Red S solution (ScienCell cat#8678a) for 30 min at RT. Dye was removed and washed 5X with diH20. Alizarin red staining was quantified by addition of 400 μL 10% acetic acid to each well. Cells and solution were collected and vortexed in 1.5 mL microcentrifuge tube for 30 s. Samples were then heated at 85° Celsisus for 10 min before incubation on ice for 5 min. Samples were next centrifuged at 20,000 g for 15 min. A concentration of 200 μL of supernatant was transferred to a new tube and 75 μL of 10% ammonium hydroxide was added to neutralize acid. Absorbance was read at 405 nm with a plate reader.
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