SaOs-2 cells or bone marrow macrophages (BMM) from wildtype were differentiated into osteoblasts. The SaOs-2 cells from human osteosarcoma cells were seeded with a cell number of 0.5x in a 24 well plate on the sample plates. 3x BMMs were seeded after isolation from murine femur and tibia bones with a cell count of cells. Cells were then treated with osteogenic medium (DMEM 0.2 mM ascorbic acid, 10 mM glycerol phosphate, 10 nM dexamethasone) for 10 days. The medium was renewed three times per week. After discarding the medium, cells were fixed with 4% PFA for 30 min and washed with distilled water (dH2O). Staining was performed with Alizarin Red S solution (2g Alizarin Red S (Sigma-Aldrich, St. Louis, MO, USA) in 100 ml dH2O at pH 4) for 45 min. De-staining was performed with 50 μl cetylpyridium chloride (Sigma-Aldrich, St. Louis, MO, USA). After 5–10 min incubation time, the solution was transferred to a 96-well microplate and the absorbance was measured at 540 nm (Infinite F200 Pro, Tecan Group AG, Maennedorf, Switzerland).
Cetylpyridium chloride
Manufactured by Merck Group
Sourced in United States, Germany
Cetylpyridium chloride is a quaternary ammonium compound used in various laboratory and industrial applications. It is a white, crystalline powder with a characteristic odor. Cetylpyridium chloride functions as a surfactant, antimicrobial agent, and disinfectant. Its chemical structure and properties make it useful in a range of laboratory and industrial processes.
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Lab products found in correlation
Alizarin red s, by Merck Group (2 mentions)
Alizarin red s solution, by Merck Group (2 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Anti runx2, by MBL Life Science (1 mentions)
Alkaline phosphatase alp assay kit, by Takara Bio (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Recombinant mouse s100a4, by ProSpec (1 mentions)
Anti β actin ac 74, by Merck Group (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Colorimetric detection kit, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Calcium detection assay kit, by Abcam (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Alizarin red s ar, by Merck Group (1 mentions)
5 protocols using cetylpyridium chloride
1
Osteoblast Differentiation and Quantification
2
Quantifying Mineralization and Osteogenic Differentiation
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Mineralization was studied using Alizarin red Staining. At day 1, 14 and 21, triplicate samples were fixed with 4% paraformaldehyde (10 min) and after washing twice with dH2O, 1 ml Alizarin red S solution (40 mM, pH 4.2, Sigma-Aldrich, A5533) was added to each well and incubated for 10 minutes at room temperature. The amount of matrix mineralization was quantified by staining the samples and dissolving the bound Alizarin red S with 10% Cetylpyridium chloride (Sigma, C0732) for 15 min at room temperature before measuring the absorption of each sample at 570 nm. Standards prepared form Alizarin S Red solutions in the concentration range 3.9 × 10−2 mM to 2,5 mM were used to produce a calibration curve.
For Von Kossa staining the cells were washed three times with PBs and fixed as above. Then 2.5% w/v silver nitrate solution was added to each sample and exposed to sunlight for 20 minutes. Then the samples were washed again with milli Q water and the unreacted silver was removed with 5% of sodium thiosulfate for 5 minutes.
For Von Kossa staining the cells were washed three times with PBs and fixed as above. Then 2.5% w/v silver nitrate solution was added to each sample and exposed to sunlight for 20 minutes. Then the samples were washed again with milli Q water and the unreacted silver was removed with 5% of sodium thiosulfate for 5 minutes.
3
Regulation of Osteoblast Differentiation
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Recombinant mouse S100A4 was purchased from Prospec (East Brunswick, NJ, USA). Phosphospecific antibodies against IκB kinase (IKK)α/β (Ser176/180), IκB (Ser32), p65 (Ser536), IKKα/β, IκB, and p65 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against osterix, laminB, and p65 were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-β-actin (AC-74) was purchased from Sigma-Aldrich (St. Louis, MO, USA), anti-Runx2 from MBL International (Woburn, MA, USA), the alkaline phosphatase (ALP) assay kit from Takara Bio Inc. (Fukui, Japan). β-Glycerophosphate, cetylpyridium chloride, and ascorbic acid from Sigma-Aldrich, Bay11-7082 from Alexis Biochemicals (Grunberg, Germany), and the Cell Counting Kit-8 (CCK-8) from Dojindo (Kumamoto, Japan).
4
Quantification of Vascular and Cellular Calcification
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Calcium content of the aortas and the kidneys was determined using the Calcium Detection Assay Kit (Abcam) following the manufacturer’s instructions and normalized to tissue weight. To quantify the calcium levels in MOVAS, cells were decalcified with HCl and the calcium content in supernatants was determined using the Calcium Detection Assay Kit (Abcam) according to the manufacturer’s instruction. Total protein was quantified using the PierceTM BCA Protein Assay Kit (Thermo Scientific) following the manufacturer’s instructions. The calcium content was normalized to the protein content.
Blood urea nitrogen (BUN) levels were measured using a colorimetric detection kit (Thermo Fisher Scientific) following the manufacturer’s instructions.
Calcium deposition was evaluated by staining the cells with Alizarin Red (Sigma Aldrich). Cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min, stained with 2% Alizarin Red for 10 min at room temperature, and rinsed with distilled water. The stained cells were extracted with 10% cetylpyridium chloride (Sigma Aldrich) for 10 min. The OD was measured at 570 nm.
Blood urea nitrogen (BUN) levels were measured using a colorimetric detection kit (Thermo Fisher Scientific) following the manufacturer’s instructions.
Calcium deposition was evaluated by staining the cells with Alizarin Red (Sigma Aldrich). Cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min, stained with 2% Alizarin Red for 10 min at room temperature, and rinsed with distilled water. The stained cells were extracted with 10% cetylpyridium chloride (Sigma Aldrich) for 10 min. The OD was measured at 570 nm.
5
Evaluating Osteogenic Differentiation of MSCs
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MSCs were seeded as described in 2.3 and cultured for 21 d in the OS medium. The presence of mineralized nodules was evaluated by alizarin red S (ARS, Sigma-Aldrich) staining [18 (link)]. After being fixed in 70% ethanol for 30 min, cells were washed with PBS and stained with ARS (40 mM, pH 4.2) for 30 min. Images were acquired using the Eclipse Ti microscope (Nikon, Tokyo, Japan) connected to a video camera. The ARS staining was eluted with 10% (w/v) cetylpyridium chloride (Sigma-Aldrich) for quantitative analysis. The absorbance was measured at 570 nm.
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