Alizarin red solution

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, France

Alizarin Red solution is a laboratory reagent used for the histological staining of calcium-containing compounds. It is a bright red dye that binds to calcium ions, allowing for the visualization of calcium deposits in various biological samples. The solution provides a simple and effective method for the detection and analysis of calcium-rich structures, such as bone, teeth, and mineralized tissues.

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Lab products found in correlation

Cetylpyridinium chloride, by Merck Group (21 mentions) Dexamethasone (dex), by Merck Group (19 mentions) Ascorbic acid, by Merck Group (18 mentions) β glycerophosphate, by Merck Group (15 mentions) Oil red o solution, by Merck Group (13 mentions) Alizarin red, by Merck Group (12 mentions) Oil red o, by Merck Group (8 mentions) Alcian blue solution, by Merck Group (6 mentions) Evos xl core, by Thermo Fisher Scientific (6 mentions) L glycerophosphate, by Merck Group (6 mentions) Alizarin red s, by Merck Group (5 mentions) Alcian blue, by Merck Group (5 mentions) Paraformaldehyde (pfa), by Merck Group (4 mentions) Synergy h1 multi mode microplate reader, by Agilent Technologies (3 mentions) Cetylpyridinium chloride monohydrate solution, by Merck Group (3 mentions) Sodium phosphate, by Merck Group (3 mentions) Cetylpyridinium chloride solution, by Merck Group (3 mentions) Spark spectrophotometer, by Tecan (3 mentions) Alcian blue 8gx, by Merck Group (3 mentions) Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (3 mentions)

208 protocols using alizarin red solution

Alizarin Red Staining for 3D Bioprinted Constructs

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The mineralization
in the 3D bioprinted constructs was analyzed using alizarin red staining.
The mono- and three-layered 3D structures were prepared by deposition
of PEG bioinks through microcapillaries with final 1 × 10⁶, 2 × 10⁶, and 5 × 10⁶ hBMSCs
densities in alternating layers. The structures were treated with
OM for 21 days. After fixation with 4% paraformaldehyde for 30 min,
the 3D bioprinted constructs were then stained with alizarin red solution
(pH: 4.1–4.2) (Sigma) at a concentration of 2 mg/mL for 1 min.
A serial washing step with distilled water was applied to remove the
excess alizarin red dye from the constructs. The monolayer hydrogel
structures were examined with a 10× objective lens under an inverted
light microscope (Carl Zeiss Microscopy). The cross sections of three-layered
gradient structures were examined under a stereomicroscope (Carl Zeiss
Microscopy).

Altunbek M., Afghah S.F., Fallah A., Acar A.A, & Koc B. (2023). Design and 3D Printing of Personalized Hybrid and Gradient Structures for Critical Size Bone Defects. ACS Applied Bio Materials, 6(5), 1873-1885.

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Alizarin Red Staining of BMSC Calcium

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The calcium deposition of BMSCs after cultured in OM for 7 d or 14 d was evaluated by Alizarin Red staining. Briefly, BMSCs were fixed in 4% paraformaldehyde for 20 min and washed by PBS for three times. Then BMSCs were stained with Alizarin red solution (Sigma Aldrich, Saint Louis, MO, USA) and photographed under a microscope (Olympus, Tokyo, Japan).

Li Y., Wang X., Pan C., Yuan H., Li X., Chen Z, & He H. (2023). Myoblast-derived exosomal Prrx2 attenuates osteoporosis via transcriptional regulation of lncRNA-MIR22HG to activate Hippo pathway. Molecular Medicine, 29, 54.

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Quantifying Osteogenic Differentiation

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Cells were cultured with CO, SBF50 or SBF100 (all media were supplemented with 10 mM ß-glycerophosphate, Sigma Aldrich, Germany) for 28 days and media were changed twice a week. Cells were washed with PBS 0.1 M and fixed with 4% paraformaldehyde in PBS. Calcium deposits were assayed by Alizarin Red S staining as followed: After air-drying, 0.5% Alizarin Red solution (pH 4, g/v) (Sigma Aldrich, Germany) was added, incubated for 10 min and subsequently the unbound dye was completely washed away. In order to the quantify Alizarin Red S signal, the dye was eluted for 15 min at room temperature using a solution of 20% methanol, 10% acetic acid and 70% distilled water and measured at 450 nm. The phosphates were stained by von Kossa dye (Sigma Aldrich, Germany) applying a 3% silver nitrate solution in the dark for 30 min, followed by three repeated washing steps and incubation with sodium carbonate-formaldehyde solution to develop the color (dark brown). The two stains were evaluated both macroscopically and microscopically; and then evaluated semi quantitatively by using the calcium- or phosphate deposition score.

Tübel J., Maier E., Jegen M., Marthen C., Obermeier A., Haug A.T., Schneider J, & Burgkart R. (2021). Patient-specific effects of soluble factors from Staphylococcus aureus and Staphylococcus epidermidis biofilms on osteogenic differentiation of primary human osteoblasts. Scientific Reports, 11, 17282.

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Isolation and Characterization of Murine and Human BMSCs

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Isolation and culture of human BMSCs from healthy volunteer donors as described previously [24 (link)]. Mouse BMSCs was isolated from tibias bone marrow of 1-month-old Mapk7^flox/flox mice. Low-glucose DMEM (Gibco, USA) supplemented with 1% penicillin–streptomycin solution (Gibco, USA) and 10% fetal bovine serum (Gibco, USA) was used as the culture medium. The culture medium supplemented with 10 mmol/L β-glycerophosphate and 50 μg/mL ascorbic acid was used as osteogenic medium. For alizarin red staining, cells were washed with PBS and fixed in 4% paraformaldehyde for 5 min, followed by being washed with PBS. Then, the cells were stained with Alizarin Red Solution (Sigma, A5533) for 10 min at room temperature. Alkaline phosphatase (ALP) staining was performed by using BCIP^®/NBT-Purple Liquid Substrate System for Membranes (Sigma, B3679) as per manufacturer’s instructions.

Yang X., Zhong D., Gao W., Liao Z., Chen Y., Zhang S., Zhou H., Su P, & Xu C. (2020). Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia. Cell & Bioscience, 10, 103.

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Quantification of Osteogenic Mineralization

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Following the 15-day culture period, the cells underwent PBS (phosphate-buffered saline) washing and fixation with 10% formaldehyde at room temperature for 15 min. Subsequently, the cells were rinsed thrice with ddH₂O and stained with a 40 mM alizarin red solution (Sigma-Aldrich, Merck Group) at room temperature for 30 min. After staining, the cells were washed four times with ddH₂O. For quantification, the stained mineralized nodules were subjected to a 30 min incubation with 10% cetylpyridinium chloride (Sigma-Aldrich). The resulting solutions were collected, and absorbance at 562 nm was measured using a Tecan Infinite M200 microplate reader. Sample quantification was performed based on the alizarin red standard curve. The inclusion of alizarin red staining, a widely accepted method for assessing calcium deposition, complemented the quantitative measures by providing a qualitative and visual evaluation of mineralization [68 (link)].

Harahap I.A., Olejnik A., Kowalska K, & Suliburska J. (2024). Effects of Daidzein, Tempeh, and a Probiotic Digested in an Artificial Gastrointestinal Tract on Calcium Deposition in Human Osteoblast-like Saos-2 Cells. International Journal of Molecular Sciences, 25(2), 1008.

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Quantifying Extracellular Matrix Mineralization

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Alizarin red staining was performed to determine the presence of extracellular matrix mineralization. After 14 days, cells were fixed in 96 % ethanol for 15 min and stained with 0.2 % alizarin red solution (Sigma Aldrich) in water (pH 6.4) at room temperature for 1 h as previously described [23 (link)]. Alizarin red staining was performed using images captured on a Nikon D610 camera with a Heerbrugg M400 ZOOM microscope (WILD HEERBRUGG, Switzerland). Image J software was used to quantify data using set parameters for colour intensity staining of red using color threshold including parameters for hue, saturation and brightness. The same threshold values were used for all analyzed. Means and standard deviations (SE) were calculated, and the statistical significance of differences was examined by one-way analysis of variance with Bonferroni test (*, p values < 0.05 was considered significant).

Fujioka-Kobayashi M., Caballé-Serrano J., Bosshardt D.D., Gruber R., Buser D, & Miron R.J. (2016). Bone conditioned media (BCM) improves osteoblast adhesion and differentiation on collagen barrier membranes. BMC Oral Health, 17, 7.

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Skeletal Preparation and Staining

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After observing the apparent phenotype of 3-week-old Prrx1-Irx3 or Irx3-Tg mice (Fig 5C), skeletal preparations were made and analyzed.
Skin and viscera were removed from the limbs of transgenic Prrx1-Irx3 or Irx3 mice. The specimens were then dehydrated in 100% ethanol for 120 h and degreased in acetone for 48 h. Subsequently, samples were stained with Alcian blue solution (7.5 g Alcian blue 8GX (Sigma) in 10 ml glacial acetic acid (Sigma) and 40 ml of 95% ethanol) at RT for 12 h. After washing with 95% ethanol for 10 min, the samples were treated with 2% KOH and stained with Alizarin red Solution (7.5 mg Alizarin red S (Sigma) in 100 ml of 1% KOH solution). Specimens were washed with 20% glycerol in 1% KOH for 120 h and then with 20% glycerol in 20% ethanol for 12 h (Fig 5D).

Yokoyama S., Furukawa S., Kitada S., Mori M., Saito T., Kawakami K., Belmonte J.C., Kawakami Y., Ito Y., Sato T, & Asahara H. (2017). Analysis of transcription factors expressed at the anterior mouse limb bud. PLoS ONE, 12(5), e0175673.

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Isolation and Characterization of Murine Bone Cells

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Calvaria cells were isolated from 3‐ to 4‐day‐old C57BL/6 mice as described.⁽³⁵⁾ To obtain bone marrow–derived stromal cells, total bone marrow cells pooled from 3–4 C57BL/6 female mice at 2–4 months of age were cultured with 20% FBS, 1% penicillin–streptomycin–glutamine, and 50 μg/ml of ascorbic acid (Sigma) in 10‐cm culture dishes for 5 days. Half of the medium was replaced every 3 days. Cells were then cultured with 10% FBS, 1% penicillin–streptomycin–glutamine, 50 μg/ml of ascorbic acid, and 10mM β‐glycerophosphate (Sigma0Aldrich) for 21 days with or without HBX. Mineralized matrix was stained with 40mM Alizarin Red solution (Sigma‐Aldrich). BrdU incorporation was measured with a Cell Proliferation ELISA Chemiluminescence Kit (Roche Diagnostics). Alkaline phosphatase activity was determined in cells cultured for 7 days, and cells were lysed in 100mM glycine, 1mM MgCl₂, and 1% Triton X‐100 at pH 10 using a buffer containing 2‐amino‐2‐methylpropanol and p‐nitrophenylphosphate (Sigma‐Aldrich). Alkaline phosphatase activity was normalized to total protein concentration, which was measured using a detergent‐compatible kit (Bio‐Rad). For all assays, cells were plated in triplicate.

Evans D.S., O'Leary M.N., Murphy R., Schmidt M., Koenig K., Presley M., Garrett B., Kim H., Han L., Academia E.C., Laye M.J., Edgar D., Zambataro C.A., Barhydt T., Dewey C.M., Mayfield J., Wilson J., Alavez S., Lucanic M., Kennedy B.K., Almeida M., Andersen J.K., Kapahi P., Lithgow G.J, & Melov S. (2021). Longitudinal Functional Study of Murine Aging: A Resource for Future Study Designs. JBMR Plus, 5(3), e10466.

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Osteogenic Differentiation and Mineralization Assays for MSCs

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To induce MSC differentiation, we cultured MSCs in mineralization-inducing media containing 100 µM ascorbic acid, 2 mM β-glycerophosphate and 10 nM dexamethasone. For ALP staining, after induction, we fixed cells with 4% paraformaldehyde and incubated them with a solution of 0.25% naphthol AS-BI phosphate and 0.75% Fast Blue BB dissolved in 0.1 M Tris buffer (pH 9.3). For detecting mineralization, we induced MSCs for 2–3 weeks, fixed the cells with 4% paraformaldehyde and stained them with 2% Alizarin Red solution (Sigma-Aldrich).
To perform TRAP staining and osteoclast quantification, we fixed cells with a mixture of 3% formaldehyde, 67% acetone and 25% citrate solution, and then stained with a TRAP kit from Sigma Aldrich according to manufacturers’ instructions. Images were taken and analyzed using an Olympus IX-51 microscope. We only counted TRAP+ multinucleated cells (>3 nuclei) as osteoclasts.

Yu B., Chang J., Liu Y., Li J., Kevork K., Al-Hezaimi K., Graves D.T., Park N.H, & Wang C.Y. (2014). Non-canonical Wnt4 prevents skeletal aging and inflammation by inhibiting NF-κB. Nature medicine, 20(9), 1009-1017.

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Osteogenic Differentiation of Cells on PLA/PCEC Scaffolds

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Cells were seeded on the hybrid PLA/PCEC scaffolds containing 50 wt% PCEC, which fixed on the bottom of 6-well plates in advance, at a density of 5×10⁵ cells/well and cultured with proliferation medium. Two days after seeding, the proliferation medium was replaced with osteogenic differentiation medium (DMEM-LG containing 10% fetal bovine serum, 1% penicillin/streptomycin, 100 nM dexamethasone, 10 mM (β-glycerophosphate, and 50 μg/mL ascorbic acid-2-phosphate). After 2 weeks of culture, specimens were rinsed with PBS twice and fixed in 10% buffered formalin, then were dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%). Subsequently, the samples were rinsed twice with hexamethyldisilazane (Kelong Chemicals, Chengdu, People’s Republic of China) for 20 minutes and left to air dry overnight in a desiccator. After vacuum drying and gold coating, the samples were viewed by SEM. For alizarin red staining, the samples were gently washed in PBS and fixed with 10% formalin for 30 minutes and then stained with alizarin red solution (Sigma-Aldrich) for 30 minutes. Calcium deposits were checked by microscope.

Wang Y., Guo G., Chen H., Gao X., Fan R., Zhang D, & Zhou L. (2014). Preparation and characterization of polylactide/poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) hybrid fibers for potential application in bone tissue engineering. International Journal of Nanomedicine, 9, 1991-2003.

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