Alizarin red s powder

Manufactured by Merck Group

Sourced in United States

Alizarin Red S powder is a chemical compound that is commonly used as a staining agent in laboratory applications. It is a red-orange crystalline powder that is soluble in water and organic solvents. The primary function of Alizarin Red S is to stain calcium-containing structures, such as bone and teeth, for visualization and analysis purposes.

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Lab products found in correlation

Paraformaldehyde (pfa), by Merck Group (2 mentions) α mem medium, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Formaldehyde solution, by Merck Group (2 mentions) Phosphate buffered saline (pbs), by Welgene (2 mentions) Cetylpyridinium chloride, by Merck Group (2 mentions) Trypsin edta solution, by Welgene (2 mentions) Axioplan 2 imaging microscope, by Zeiss (1 mentions) Ammonium hydroxide, by Merck Group (1 mentions) Te2000 u, by Nikon (1 mentions) Bisbenzimide h 33342, by Merck Group (1 mentions) Eosin solution, by Thermo Fisher Scientific (1 mentions) Nanozoomer digital pathology system, by Hamamatsu Photonics (1 mentions) Ab150687, by Abcam (1 mentions) Gill 2 hematoxylin solution, by Leica (1 mentions) Tissue tek o c t freezing compound, by Sakura Finetek (1 mentions) Decalcifying solution, by Merck Group (1 mentions) Antibiotic antimycotic solution, by Merck Group (1 mentions) Rat fgf 2 elisa kit, by R&D Systems (1 mentions) Rat osteoblast differentiation medium, by Cell Applications (1 mentions)

Close spelling variants of this product

Alizarin Red S (1280 citations) Alizarin Red (866 citations) S powder (11 citations) Alizarin Red powder (7 citations) Alizarin Powder (3 citations)

9 protocols using alizarin red s powder

Assessing Scaffold Architecture and Mineralization

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For general scaffold architecture assessment and cell distribution, a standard haematoxylin and eosin (H&E) staining (Waldeck GmbH & Co. KG, Münster, Germany) was performed on the sections, followed by Masson’s trichrome staining for collagen fibres (Merck KGaA).
To detect newly formed HA deposits within the scaffolds, Alizarin Red staining was performed. For the preparation of the dye, 2 g Alizarin Red S powder (Sigma-Aldrich) was dissolved in 100 ml Aqua Dest. Next, the pH was adjusted to 7.2 by adding ammonium hydroxide (Sigma-Aldrich). The slides were placed in the solution for 120 seconds and the excess dye was washed off. Using a microscope (Axioplan 2 Imaging Microscope; Carl Zeiss AG, Oberkochen, Germany) with a 5× and 10× magnification, sections of all 12 scaffolds were examined and classified by a ranking system. Each scaffold was graded according to the amount of Alizarin Red stain: ‘0’ = 0% stained area; + = 1% to 25% stained area; ++ = 26% to 50% stained area; +++ = 51% to 75% stained area; and ++++ = 76% to 100% stained area.

Grossner T.L., Haberkorn U, & Gotterbarm T. (2019). 99mTc-Hydroxydiphosphonate quantification of extracellular matrix mineralization in 3D human mesenchymal stem cell cultures. Bone & Joint Research, 8(7), 333-341.

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Alizarin Red S and Immunohistochemistry for Osteogenic Differentiation

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Alizarin Red S staining was performed at day 21 according to an established protocol (PromoCell, Heidelberg, Germany). The stain solution was made from Alizarin Red S powder (Sigma-Aldrich, St. Louis MO). The cells were fixed inside the warmed gels and the stain was allowed to permeate gels while excess stain was subsequently rinsed out with several washes of deionized water. Mineralization was visualized with light microscopy. Images were processed with ImageJ and quantified via threshold intensity.
Osteopontin (OPN) and osteocalcin (OCN) expression were assessed via immunohistochemistry at day 21. The primary antibody was prepared as 1:200 dilution in 0.05% BSA/PBS. The secondary antibody was prepared as a 1:500 dilution in 0.05% BSA/PBS. The nuclear counterstain used was bisBenzimide H 33342 (Sigma-Aldrich, St. Louis MO). Fluorescent images were taken by Nikon TE-2000U. 4–5 images were taken per group and percentage of positive expression was quantified as percentage of positively staining cells over total cells. Cells with a positive signal for the stain were identified by comparing experimental wells to control wells to which only secondary antibody was added, omitting primary. Cells that were positive for OPN and OCN were quantified via histomorphometry.

Morochnik S., Zhu Y., Duan C., Cai M., Reid R.R., He T.C., Koh J., Szleifer I, & Ameer G.A. (2018). A Thermoresponsive, Citrate-based Macromolecule for Bone Regenerative Engineering. Journal of biomedical materials research. Part A, 106(6), 1743-1752.

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Histological Analysis of Frozen Tissue Samples

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After micro-CT analysis, each of the three samples were rinsed in PBS, embedded in Tissue-Tek ® O.C.T. freezing compound (Sakura Finetek, Netherlands), then stored at −80°C. 14 μm thick histology specimens were cut from frozen block using a motorized Microm HM 550 cryostat (Thermo Fisher Scientific) and placed onto glass slides. Samples were H&E stained using Gill II Hematoxylin solution (Leica, Wetzlar, Germany) and with a Eosin solution made from Eosin Y powder (Fisher Scientific, Pittsburg, PA), and were Alizarin Red stained with a solution made from Alizarin Red S powder (Sigma-Aldrich), and Von Kossa stained using a kit (ab150687, Abcam, Cambridge, UK), then imaged using a NanoZoomer Digital Pathology System (Hamamatsu, Japan). All images taken were analyzed qualitatively.

Dewey M.J., Johnson E.M., Weisgerber D.W., Wheeler M.B, & Harley B.A. (2019). Shape-fitting collagen-PLA composite promotes osteogenic differentiation of porcine adipose stem cells. Journal of the mechanical behavior of biomedical materials, 95, 21-33.

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Alizarin Red S Staining for Mineralization

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After 21 days, cell/scaffold constructs were washed thrice in phosphate-buffered saline (PBS) and fixed for 30 min in 4% paraformaldehyde (Merck & Co, White House Station, NJ, USA), followed by 2% Alizarin red S powder (Sigma-Aldrich) dissolved in distilled water and pH adjusted to 4.2. Samples were stained for 20 min and imaged with a light microscope. For quantification of Alizarin red stain, 600 µL of 100 µM cetylpyridinium chloride was added into cell/scaffold construct and left to shake overnight. The stain was diluted 1:3 with cetylpyridinium chloride, and the absorbance measured at 540 nm.

Munir A., Døskeland A., Avery S.J., Fuoco T., Mohamed-Ahmed S., Lygre H., Finne-Wistrand A., Sloan A.J., Waddington R.J., Mustafa K, & Suliman S. (2019). Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration. Journal of Tissue Engineering, 10, 2041731419852703.

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Quantitative Alizarin Red S Analysis

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To analyze the calcium deposition, Alizarin Red S staining was performed. Cells were incubated with OM for 14 days, then the cells were fixed with 10% (v / v) formalin and washed three times with PBS. To make the ARS solution, 20 mg of Alizarin Red S powder (Sigma-Aldrich, USA) was dissolved in 1 ml of distilled water and the pH was adjusted to 4.1 ~ 4.2 with ammonium hydroxide (NH₄OH). Fixed cells were stained with ARS solution for 20 min and washed 3 times with distilled water for 5 min. For ARS quantification, 800 μl of 10% (v / v) acetic acid per well was added and incubated at room temperature for 30 min. The cells were collected with a cell scraper, transferred to a 1.5 ml tube, and 500 μl of mineral oil was added. The samples were heated at 85 °C for 10 min and cooled with ice for 5 min. The solution was then centrifuged at 20,000 G for 15 min. After centrifugation, 500 μl of supernatant was collected. Then, 200 μl of 10% ammonium hydroxide was added to the supernatant to complete precipitation. To see the results, Absorbance values were measured with a spectrometer.

Kim J., Kim H.D., Park J., Lee E.S., Kim E., Lee S.S., Yang J.K., Lee Y.S, & Hwang N.S. (2018). Enhanced osteogenic commitment of murine mesenchymal stem cells on graphene oxide substrate. Biomaterials Research, 22, 1.

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Alizarin Red Staining Protocol

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Alizarin Red solutions were prepared by dissolving 1 g Alizarin Red S powder (Sigma-Aldrich) in 50 mL DI water. After the pH of the solution was adjusted just below 4.2 with hydrochloric acid, the solution was filtered using a 0.2 μm filter and stored in the dark. Samples were washed twice with PBS and fixed with 4% PFA for 10 min. Samples were washed three times with DI water, then incubated with the Alizarin Red working solution. After 1 h incubation, samples were washed six times with DI water and imaged by bright field microscopy.

Peng L, & Gautrot J.E. (2021). Long term expansion profile of mesenchymal stromal cells at protein nanosheet-stabilised bioemulsions for next generation cell culture microcarriers. Materials Today Bio, 12, 100159.

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In Vitro Osteoblast Differentiation

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Alizarin Red S powder, formaldehyde solution, antibiotic-antimycotic solution, decalcifying solution, and cetylpyridinium chloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). α-MEM medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Rat osteoblast differentiation medium was provided by Cell Applications (San Diego, CA, USA). Phosphate-buffered saline (PBS) and 0.25 % trypsin-EDTA solution were purchased from Welgene (Daegu, Korea). Primary antibodies against osterix, alkaline phosphatase (ALP), and GAPDH were purchased from Abcam (Cambridge, UK). Type-1 collagen and FGF2 were obtained from Novus Biologicals (Centennial, CO, USA). Antibodies to P-Met, c-Met, and Runx-2 were provided by Cell Signaling Technology (Danvers, MA, USA). The osteocalcin antibody was obtained from Bioss (Woburn, MA, USA). Hydroxyapatite/beta-tricalcium phosphate (HA/β-TCP) ceramic powder was purchased from Biomatlant (Vigneux, France). Rat HGF ELISA Kit, Rat FGF2 ELISA Kit and recombinant FGF2 protein were obtained from R&D Systems (Minneapolis, MN, USA). The rat VEGF enzyme-linked immunosorbent assay (ELISA) kit was purchased from Abclonal (Woburn, MA, USA). Rat SDF-1α ELISA Kit was provided by Biorbyt (Cambridge, UK). Recombinant rat HGF protein was purchased from LifeSpan BioSciences (Seattle, WA, USA).

Park J.S., Kim D.Y, & Hong H.S. (2024). FGF2/HGF priming facilitates adipose-derived stem cell-mediated bone formation in osteoporotic defects. Heliyon, 10(2), e24554.

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Isolation and Characterization of ADSCs

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Healthy adipose tissues were provided by the Kyung Hee University Medical Center [Total of eight donors (6 male, 2 female); Seoul, Korea; (IRB# 2016-12-022)] with donors' written consent. Adipose tissue (2×2 cm) was suspended in saline in a 50 ml tube at 4°C and transferred to the cell culture room. ADSC isolation was performed immediately upon arrival to the culture room. ADSCs from healthy young individuals were purchased from ScienCell Research Laboratories (individuals <30 years old). Alizarin Red S powder, Oil Red O solution, formaldehyde solution, and cetylpyridinium chloride were purchased from Sigma-Aldrich; Merck KGaA. Stempro osteogenesis and adipogenesis kits, α-MEM medium, and FBS were purchased from Gibco; Thermo Fisher Scientific, Inc. Phosphate-buffered saline, penicillin/streptomycin, and 0.25% trypsin-EDTA solution were provided by Welgene. Vascular endothelial growth factor (VEGF), hepatocyte grow factor (HGF), stromal cell-derived factor-α (SDF-1α), transforming growth factor-β1 (TGF-β1), and the bone morphogenetic protein (BMP)-2 ELISA kits were obtained from R&D Systems.

Park J.S., Park G, & Hong H.S. (2020). Age affects the paracrine activity and differentiation potential of human adipose-derived stem cells. Molecular Medicine Reports, 23(2), 160.

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Osteocyte Differentiation Protocol

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Cells were seeded at 6x10 3 cells per cm 2 in a 24 well plate. After 4 days of culture the media was removed and replaced with 500µl/well osteogenesis differentiation medium (Osteocyte Differentiation Basal Medium supplemented with 10% Osteogenesis supplement, 1% PSF; Life Technologies). The media was replaced every 3 to 4 days. On day 25 the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with Alizarin Red S staining solution (2g Alizarin Red S powder (Sigma-Aldrich) in 100ml distilled water and the pH was adjusted to 4.2).

Davies L.B., Jones R.H, & Thornton C.A. (2019). Maternal Serum, an Isolation and Expansion Tool for Umbilical Cord Matrix Mesenchymal Stromal Cells. Tissue engineering. Part C, Methods, 25(4).

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