Rat BMSCs were cultivated in 24-well plates (1×104 cells/well) for 21 days. After 30 minutes of paraformaldehyde fixation (4%, Meilunbio), the cells were then stained with 2% Alizarin Red S (Sigma) at room temperature for 20 minutes. Quantification was performed by treating the stained calcium nodules with 10% cetylpyridinium chloride solution (Sigma). Microplate readers (Bio-Rad) were used to measure the absorbance at 570 nm.
Cetylpyridinium chloride solution
Manufactured by Merck Group
Sourced in United States
Cetylpyridinium chloride solution is a chemical compound that consists of a pyridinium cation and a chloride anion. It is a colorless, viscous liquid that is soluble in water and other polar solvents. The core function of this solution is as a surfactant and antimicrobial agent.
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Lab products found in correlation
Alizarin red s, by Merck Group (5 mentions)
Alizarin red s solution, by Merck Group (5 mentions)
β glycerophosphate, by Merck Group (5 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Alizarin red solution, by Merck Group (3 mentions)
Alizarin red, by Merck Group (3 mentions)
Ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Alizarin red s ar, by Merck Group (2 mentions)
L ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
L ascorbic acid, by Merck Group (2 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Iscript rt supermix, by Bio-Rad (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
Alizarin red staining solution, by Solarbio (1 mentions)
31 protocols using cetylpyridinium chloride solution
1
Rat BMSC Mineralization Assay
2
Quantifying Odontoblast Mineralization
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After 14 days of odontoblastic differentiation, the cells were incubated with 2% alizarin red staining solution (Beyotime Biotechnology, Haimen, China) for 10 min at room temperature. Then, the cells were observed under an inverted microscope and the cell mineralization was quantified with alizarin red extracted with 100 mM cetylpyridinium chloride solution (Sigma, St Louis, MO, USA), and the OD value was read at 570 nm.
3
Extracellular Matrix Mineralization Analysis
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Further, remineralization ability has been performed in direct contact cells using alizarin red S which targets calcium deposits in the extracellular matrix. After 14 days, 40 mM of alizarin red S solution (pH 4.2) was added to the 24-well plates and incubated for 30 min. Then, quantitative calcium analysis (semi-quantification) of mineralized matrix nodules generated from the cells was performed by adding 10% cetylpyridinium chloride solution (Sigma-Aldrich). The optical density values were read at 560 nm, which represented the relative quantity of mineralization nodules.
4
Alizarin Red Staining of PDLSCs
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PDLSCs were fixed in 70% ethanol for 1 h and washed with deionized water. We added 40 mM alizarin red staining solution (pH 4.2) into the 6-well plates, incubated the cells at room temperature for 10 min, washed the cells with deionized water 5 times, viewed them under a microscope, and captured the images. For quantitative calcium analysis, the cells were treated with 10% cetylpyridinium chloride solution (Sigma-Aldrich) for 30 min at room temperature. The OD562 was used to quantify the degree of mineralization and calcium quantitative analysis for alizarin red staining was normalized to the total protein content before calculation. The experiments were repeated at least 3 times.
5
Quantitative Alizarin Red S Assay for Mineralization
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The cells were placed in a 24-well plate at a density of 1 × 105 cells per well and cultured for 24 h. Then, the medium was switched to material extract for the duration of the experiment. After exposure to the extract medium for 14 days, mineralization was assessed by staining with Alizarin red S (Sigma-Aldrich). In brief, 40 mmol/L of Alizarin red S was prepared in distilled water, adjusted to a pH of 4.2 with ammonium hydroxide, and then applied to the cells for 10 min at room temperature with gentle agitation. After being washed with de-ionized water, the stained cell culture plate was moved to a scanner, and the stained image was acquired. For quantitative evaluation, the sample was reacted with 10% cetylpyridinium chloride solution (pH 7.0; Sigma-Aldrich) at room temperature for 15 min to dissolve the stain, and then absorbance was measured at a wavelength of 540 nm with a standard solution.
6
Alizarin Red S Staining for Osteogenic Differentiation
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The hDPCs were distributed in a 24-well culture plate at a density of 2×104 and cultured for 24 h. Then, the cells were treated with different concentrations of ECN for the duration of the experiment. After the hDPCs were cultured for 14, 21, and 28 days, the cells were washed with PBS and reacted with 40 mmol/L of alizarin red S solution (Sigma-Aldrich) adjusted to a pH of 4.2 with ammonium hydroxide. After being washed with distilled water, the samples were reacted with 10% cetylpyridinium chloride solution (Sigma-Aldrich) at room temperature for 15 min to dissolve the stain. The contents from each well were transferred to a 96-well plate and were measured spectrophotometrically in a microplate reader (SPECTROstar Nano).
7
Osteogenic Differentiation of Mesenchymal Stem Cells
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After expansion, MSC were subjected to osteogenic-induction medium consisting of high-glucose DMEM, 10% FCS (Biochrom, Berlin, Germany), 0.1 μm dexamethasone, 0.17 mM ascorbic acid 2-phosphate, 10 mM β-glycerophosphate (all from Sigma, Deisenhofen, Germany), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured for 3 weeks with medium changes twice per week. To monitor osteogenic differentiation, cells were stained with 0.5% Alizarin Red S (Chroma, Münster, Germany), to detect calcium deposition. Next, they were treated with 10% cetylpyridiniumchloride-solution (Sigma, Deisenhofen, Germany) to extract calcium-bound dye, and calcium content was measured using spectrophotometry at 570 nm. The values were normalized to the total protein content in cell lysates using Bradford Reagent (Sigma, Deisenhofen, Germany).
8
Evaluating Dental Pulp Stem Cell Mineralization
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Expression of osteo/odontoblastic-related genes was performed with qRT-PCR after exposing the DPSCs to the MTA extracts for 7 and 14 days (Figure 1 ). RNA extraction was carried out with the RNA extraction kit (PureLink™ RNA Mini Kit, LifeTech, Carlsbad, California. USA), followed by cDNA synthesis (iScript RT Supermix, Bio-Rad, Hercules, California, USA) and PCR (Biorad-CFX Connect™ Real time system, Hercules, California, USA). The primer sequences are listed in Figure 1 . DPSC in culture medium alone served as control. Alizarin red staining (ARS) was performed to detect mineralization after 14 days. The cells were washed with PBS and fixed for 30 minutes. Alizarin red solution (Sigma-Aldrich, Saint Louis, Missouri, USA) was added and incubated for 30 minutes at 37°C. Ten percent of cetylpyridinium chloride solution (Sigma-Aldrich) was added to each well, and the optical density was measured by a plate reader (wavelength 540 nm). The culture medium was the negative control, and the osteogenic induction medium was included as a positive control. Tests were performed in triplicates.
Primer sequences ![]()
9
Alizarin Red Staining for Osteoblast Differentiation
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Cells were induced to osteoblast differentiation for 12 days. Cells were fixed with 70% ice-cold ethanol for 1 h at − 20 °C, and stained with 40 mM Alizarin red S (AR-S; Sigma-Aldrich), pH 4.2 for 10 min at room temperature. Alizarin red stain was eluted using 10% (w/v) cetylpyridinium chloride solution (Sigma-Aldrich) with shaking for 20 min and the absorbance of the eluted dye was measured at 570 nm.
10
Ozonized Water Disinfection Protocol
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Ozonized water was generated from TW using a small generator, OZS-PTDX (Sakuragawa Pump, Osaka, Japan). The ozonized water generators generate ozone by surface discharge control technology [21] and mix ozone into the water using the ejector system. TW (Water Works Bureau at Okayama City, Japan) and phosphate-buffered saline (PBS: Gibco, USA, pH 7.4) were used as negative controls. Aqueous 0.1 % cetylpyridinium chloride solution (CPC: Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control.
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