Primary osteoblast precursor cells were isolated from neonatal mouse calvaria by digestion with 0.1% collagenase (Life Technologies, Carlsbad, CA, USA) and 0.2% dispase II (Roche Diagnostics GmbH, Mannheim, Germany). Isolated osteoblast precursor cells were cultured in α-Minimal Essential Medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. For osteoblast differentiation, primary osteoblast precursor cells were cultured in osteogenic medium containing BMP2 (100 ng/mL), ascorbic acid (50 µg/mL), and β-glycerophosphate (100 mM) for six to nine days. Cultured cells were fixed with 70% ethanol and stained with 40 mM alizarin red (pH 4.2). After nonspecific staining was removed with phosphate-buffered saline, alizarin red staining was visualized with a CanoScan 4400F (Canon Inc., Tokyo, Japan). Alizarin red-stained cells were dissolved with 10% cetylpyridinium (Sigma-Aldrich), and absorbance of the extracted solution was measured at 562 nm for quantification.
Cetylpyridinium
Manufactured by Merck Group
Sourced in United States
Cetylpyridinium is a chemical compound used as a preservative and antimicrobial agent in various lab equipment and solutions. It functions as a cationic surfactant, possessing the ability to disrupt cell membranes and inhibit the growth of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Roche (2 mentions)
Alizarin red, by Merck Group (2 mentions)
P nitrophenyl phosphate substrate, by Merck Group (2 mentions)
Canoscan 4400f, by Canon (1 mentions)
Chondroitin sulphate, by Merck Group (1 mentions)
1 9 dimethylmethylene blue dmb binding, by Merck Group (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Alizarin red solution, by Merck Group (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
Oil red, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
7 protocols using cetylpyridinium
1
Isolation and Differentiation of Primary Osteoblast Precursors
2
Quantitative Analysis of Extracellular Matrix and Cell Differentiation
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Transwell discs were stained as described (Barter et al., 2015 (link)). Chondrogenic pellets and transwell discs were digested with papain (10 U/ml) at 60°C (Murdoch et al., 2007 (link)). The sulphated glycosaminoglycan (GAG) content was measured by 1,9-dimethylmethylene blue (DMB) binding (Sigma) using chondroitin sulphate (Sigma) as standard (Farndale et al., 1982 (link)), and the DNA content was measured with PicoGreen (Invitrogen) intercalating dye following the manufacturer's instructions. Cells undergoing osteoblast differentiation were fixed in 70% cold ethanol (5 min, −20°C). After drying the wells to reveal calcium-rich mineralisation deposits, the cells were incubated at room temperature with a solution of Alizarin Red (Sigma) (40 mM, pH 4.2) for 20-30 min. For quantification the staining was extracted with 10% (w/v) cetylpyridinium (Sigma) solubilised in 10 mM sodium phosphate buffer (pH 7) and the absorbance measured at 620 nM. Cells undergoing adipogenesis were fixed with formalin for 1 h, washed with distilled water and 60% isopropanol then dried. To reveal the presence of lipid droplets, the cells were stained with a 21% (w/v) solution of Oil Red O for 10 min. For quantification the staining was extracted with 100% isopropanol and the absorbance measured at 500 nM. Stained cells were washed with distilled water prior to image acquisition.
3
Isolation and Characterization of Primary Osteoblasts
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Primary osteoblasts were isolated from neonatal mouse calvaria by successive enzymatic digestion with 0.1% collagenase (Life Technologies) and 0.2% dispase II (Roche Diagnostics GmbH, Germany). Osteoblasts were cultured in osteogenic medium containing BMP2 (100 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (100 mM). To assess their differentiation, osteoblasts cultured for 3 days were stained for alkaline phosphatase (ALP). To assess ALP activity, cells were lysed in osteoblast lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA], and the lysates were incubated with p-nitrophenyl phosphate substrate (Sigma-Aldrich). ALP activity was then measured at an absorbance of 405 nm. To assess their function, osteoblasts cultured for 9 days were fixed with 70% ethanol and stained with 40 mM alizarin red (pH 4.2). For alizarin red solution assay, stained alizarin red was dissolved with 10% cetylpyridinium (Sigma) and the extracted solution was then measured at an absorbance of 562 nm.
4
Quantitative Calcium Deposition Assay
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Media was removed from the plates and these were gently washed with phosphate-buffered saline (PBS). Calcium deposits on the plate were stained with a 1% solution of Alizarin red, which was then washed with water until residual unbound dye was removed. The plates were then imaged using a plate scanner. Quantification of total calcium in each well was by immersing the alizarin-stained samples in 5% cetylpyridinium chloride (Sigma Aldrich) solution for 2 h, yielding a purple destain solution containing the solubilised cetylpyridinium–alizarin complex, which was quantified in a spectrophotometer at 562 nm.
5
Osteogenic Differentiation of hMSCs
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Human MSCs were cultured and transfected with either miCon or miR-324-5p in 96 well plates as described above. After 48 h media was replaced with OsteoMax medium (Merck Millipore) in the presence or absence of Recombinant Indian Hedgehog (Ihh) (2 μg/ml; R&D systems) and BMP2 (100 ng/ml; R&D systems). Fresh OsteoMax medium was added after 4 days. After 7 days cells were fixed in 10% formalin and mineralisation assessed by incubation with Alizarin Red solution (Sigma) (40 mM, pH 4.2) for 30 min. For quantification the staining was extracted with 10% (w/v) cetylpyridinium (Sigma) solubilised in 10 mM sodium phosphate buffer (pH 7) and the absorbance measured at 620 nM.
6
Adipogenic Differentiation Assay
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IGF-1 was purchased from Peprotech (Rocky Hill, NJ, USA). DMEM, Foetal bovine serum, β-Glycerol Phosphate, dexamethasone, ascorbic acid 2-phosphate, insulin, 3-Isobutyl-1-methylxanthine, indomethacin, rosiglitazone, Cetylpyridinium, Oil Red, Alizarin Red were purchased from Sigma-Aldrich (St. Louise, MO, USA). Other products were also purchased from Sigma-Aldrich unless otherwise indicated.
7
Osteoblast Differentiation and ALP Activity Assessment
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Mouse bone marrow stromal cells were isolated by flushing the femurs and tibiae from 6-week-old male ICR mice, and the isolated cells were cultured in α-MEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Osteoblast differentiation was induced by incubating the cells in an osteogenic medium containing 50 ng/ml IGF-1, 50 μg/ml ascorbic acid, and 100 μM β-glycerophosphate for 4 to 9 days; the culture medium was replaced every 4 days for the ALP activity assay. The osteoblast precursor cells were lysed using the osteoblast lysis buffer (50 mM NaCl [pH 7.6], 150 mM NaCl, 0.1% Triton X-100, and 1 mM EDTA). The cell lysates were incubated with p-nitrophenyl phosphate substrate (Sigma-Aldrich), and ALP activity was measured using a spectrophotometer at 405 nm. For alizarin red staining, the cells were cultured for 9 days, and were fixed with 70% ethanol and stained with 40 mM alizarin red (pH 4.2). The nonspecific staining was removed by phosphate-buffered saline (PBS) wash, and alizarin red staining was visualized with a CanoScan 4400F scanner (Canon, Japan). Alizarin red was then dissolved using 10% Cetylpyridinium (Sigma-Aldrich) for 15 min at room temperature, and alizarin red activity was measured using a spectrophotometer at 562 nm.
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