Osteoblast maturation was determined by evaluating cell mineralization using the alizarin red S dye staining. Osteoblasts were seeded in 6 cm tissue culture dishes coated with chitosan nanofiber scaffolds, and were treated with a combination of 10 nM dexamethasone, 100 μg/mL ascorbic acid, and 10 mM β-glycerophosphate for 21 days. After drug treatment, osteoblasts were washed with ice-cold PBS and then fixed in ice-cold 10% formalin for 20 minutes. Cells were washed with PBS and photographed using phase-contrast microscopy. The reaction was stopped by washing cells twice with deionized water. For the alizarin red S dye protocol, fixed osteoblasts were thoroughly rinsed and then incubated in 1% Alcian blue pH 2.5 (Thermo Fisher Scientific) for 12 hours. Sections were then incubated in alizarin red S (Thermo Fisher Scientific) for 8 minutes, dehydrated briefly in xylene, and covered with a coverslip in Permount (Thermo Fisher Scientific). Mineralized nodules were visualized and counted using an inverted microscope. Each experiment was performed in duplicate wells and repeated three times.
Alizarin red s
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alizarin Red S is a chemical compound commonly used as a histological stain in the field of biology and biochemistry. It is a red-orange powder that reacts with calcium ions to produce a bright red color. This property makes Alizarin Red S a useful tool for the detection and visualization of calcium-containing structures in various samples.
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Lab products found in correlation
Alizarin red s, by Merck Group (7 mentions)
β glycerophosphate, by Merck Group (3 mentions)
Pezgs0416, by Merck Group (2 mentions)
Spectramax id3, by Molecular Devices (2 mentions)
24 well plate, by NEST Biotechnology (2 mentions)
Acetic acid, by Thermo Fisher Scientific (2 mentions)
Ammonium hydroxide, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
Primovert, by Zeiss (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Evos m5000, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid phosphate, by Merck Group (1 mentions)
Alizarin red s, by Agilent Technologies (1 mentions)
Vectamount permanent mounting medium, by Vector Laboratories (1 mentions)
Alcian blue, by Polysciences (1 mentions)
Alizarin red s, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Cm3050s cryostat, by Leica camera (1 mentions)
30 protocols using alizarin red s
1
Osteoblast Mineralization Assay Using Alizarin Red
2
Mineralization of Rat Calvarial Osteoblasts
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Mineralization of rat calvarial osteoblasts was examined using the alizarin red S dye-staining protocol as described previously [45 (link)]. Rat calvarial osteoblasts were seeded in 6-cm tissue culture dishes overnight. Osteoblasts were then treated with 100 μM MPP, a differentiation reagent (10 nM dexamethasone, 100 μg/mL ascorbic acid, and 10 mM β-glycerophosphate), a differentiation reagent + MPP, a differentiation reagent + estradiol, and a combination of a differentiation reagent + MPP + estradiol for 21 days. The differentiation reagent, MPP, and estradiol were renewed every 2 days. After drug treatment, the cell morphology was observed and photographed using a microscope (Nikon). Also, rat calvarial osteoblasts were washed with ice-cold PBS and then fixed in ice-cold 10% formalin for 20 min. For the alizarin red S dye protocol, fixed osteoblasts were thoroughly rinsed and then incubated in 1% alcian blue at pH 2.5 (ThermoFisher Scientific, Tewksbury, MA, USA) for 12 h. Sections were then incubated in alizarin red S (ThermoFisher Scientific) for 8 min, dehydrated briefly in xylene, and covered with a coverslip in Permount (ThermoFisher Scientific). Mineralized nodules were counted and statistically analyzed.
3
Quantifying Osteogenic Mineralization in BMSCs
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BMSCs were fixed with 70% ethanol and then stained with 2% Alizarin Red S (MilliporeSigma). After washing in phosphate-buffered saline (PBS), the images of stained cells were visualized under a light microscope (Leica Microsystems, Germany). For quantification of mineralization, cells were incubated for one hour with 1 ml of cetylpyridinium chloride buffer to extract Alizarin Red S. Afterwards, the level of Alizarin Red S in total protein was determined by monitoring the absorbance at 562 nm via a microplate reader (Thermo Fisher Scientific). Alizarin Red S accumulation was expressed as μmol/μg protein.
4
Isolation and Quantification of TiO2 Nanoparticles
5
Osteogenic Differentiation of Mesenchymal Stem Cells
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MSCs were differentiated into osteogenic lineages, for which cells (P3) were seeded in triplicate, one as a negative control, onto 12-well plates at a density of 2 × 104 cells/well. After 48 h, the medium was switched to an inducing medium (StemPro Osteogenesis Differentiation Kit) or maintained in regular growth medium for a negative control sample. After 21 days, cells were fixed in 4% paraformaldehyde and Ca2+ deposits stained with 1% Alizarin Red S (Acros Organics, New Jersey, United States). The plates were analyzed using an inverted microscope (EVOS M5000, Invitrogen).
6
Dental Mesenchyme Culture and Odontogenic Differentiation
7
Osteogenic Differentiation with XAV939
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A total of 2 × 105 tdTomato + cells were cultured in a 24-well plate and induced in osteogenic differentiation medium supplemented with 10 nM dexamethasone, 100 µM L-ascorbic acid phosphate, and 5 mM β-glycerophosphate (Sigma-Aldrich). XAV939 was added to the culture media of different groups. After 21 days of induction, mineralized nodules were detected by staining with 2% alizarin red S (400480250, ACROS Organics, Fair Lawn, NJ, United States). alizarin red S crystals were dissolved in distilled water with 10% cetylpyridinium chloride and measured at 590 nm on a BioTek ELx808 system (BioTek Instruments, Vermont, United States).
8
Dental Mesenchyme Culture and Odontogenic Differentiation
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The dental mesenchyme was obtained from 1-month old Runx2fl/fl and Gli1-CreERT2;Runx2fl/fl mice 5 days after induction as described above. The pulp tissue was cut into pieces and incubated with growth media in 6-well plate. The culture media was left to rest for 4 days to allow tissue adhesion and cell migration. Then the culture media was changed every other day.
2 × 105 MSCs were plated in a 4-well chamber slide (PEZGS0416, Millipore) or 24-well plate (Nest, 702011) and induced in odontogenic differentiation media containing 1% FBS, β-glycerophosphate (β-GP) (5mM), ascorbic acid (50μg/ml), and dexamethasone (DEX) (10nM). 100 ng/ml mouse recombinant Igf2 was added to odontogenic induce media according different groups (showed inFigures 6F –6I ). For Dspp RNAscope in situ hybridization, cells cultured in a 4-well chamber slide were harvested 7 days after induction and the standard protocols of the manufacturer were then followed. To detect mineralized nodules, cells were induced for 21 days, fixed with 4% paraformaldehyde, and stained with 2% Alizarin red S (pH4.2) (ACROS Organics, 400480250). Alizarin red-S contents were extracted with 10% cetylpyridinium chloride in distilled water and measured at 590 nm on SpectraMax iD3 (Molecular Devices, LLC., San Jose, USA).
2 × 105 MSCs were plated in a 4-well chamber slide (PEZGS0416, Millipore) or 24-well plate (Nest, 702011) and induced in odontogenic differentiation media containing 1% FBS, β-glycerophosphate (β-GP) (5mM), ascorbic acid (50μg/ml), and dexamethasone (DEX) (10nM). 100 ng/ml mouse recombinant Igf2 was added to odontogenic induce media according different groups (showed in
9
Alizarin Red S Staining of Murine Hearts
10
Vertebral Anatomy Quantification Across Populations
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We counted the number of vertebrae in 721 (328 adults and 393 juveniles) individuals originating from six subpopulations and representing both wild-caught and laboratory reared individuals (Fig. 1a ; Table 1 ). In adults, we counted the vertebrae by analysing radiographs (generated by the x-ray system Arcoma PanoRad, Mediel accessed at the County Hospital of Kalmar, Sweden) in the software program ImageJ and/or by dissection after all soft tissues were removed by boiling (Fig. 1b ). In juveniles, vertebrae were visualized by staining the vertebral column using Alizarin red S (Acros Organics, New jersey, USA) according to a method described by Connolly and Yelick70 (link) followed by dissection. Specimens were then photographed in a dissection microscope (Olympus Digital camera UC 30, Olympus Microscope SZX12) and vertebrae were counted by image analysis using ImageJ software (Fig. 1c ). To determine the repeatability of VN counts, 69 individuals (Nadults = 39, Njuveniles = 30) were counted twice and blind with respect to the first count. Measurement repeatability (intraclass correlation coefficient)71 was very high for both life stages (adults: 99.1%, F38,39 = 114.16, P < 0.001; juveniles: 96.7%, F29,30 = 31.71, P < 0.001), with less than 1% and 4% of the variance in VN being due to measurement error in adults and juveniles, respectively.
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