Alizarin Red S  (Synonyms: ARS)

Alizarin Red S (ARS) is an anthraquinone derivative dye. When combined with cations such as calcium ions, the functional group of Alizarin Red S can form a coordination bond with the cation through the oxygen atom to show orange-red fluorescence. Alizarin Red S can be used for screening of calcium compounds in synovial fluid and detecting osteoblast differentiation, and can also be used for bone staining in mice. Excitation/emission wavelength: 500/570 nm^{[1][2][3][4][5]}.

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Preparation of Alizarin Red S^[1]
1.1 Prepare a 2% aqueous solution of Alizarin Red S sodium.
1.2 Use a pH meter to add ammonium hydroxide dropwise while stirring to adjust the pH to 4.2.
1.3 Filter the solution through a 0.45 μm microfilter and store in an amber bottle.
1.4 Refilter the solution through a 0.22 μm microfilter immediately before use.
2. Alizarin Red S detection of osteoblast differentiation^[2]
2.1 Osteogenic differentiation induction: MC3T3-E1 cells are seeded in well plates and are cultured in α-MEM (without ascorbic acid) supplemented with 10% fetal bovine serum. Osteogenic differentiation is induced by osteogenic medium (OSM) containing ascorbic acid (50 μg/mL) and β-glycerophosphate (10 mM).
2.2 Staining: MC3T3-E1 cells are washed with 1×PBS, fixed with 70% ethanol at room temperature for 10 min, rinsed with distilled water, and then stained with Alizarin Red S for 10 min to detect calcium deposition in the matrix.
2.3 Data analysis: The area of the red region is positively proportional to the degree of osteogenic differentiation.
3. Alizarin Red S Screening test for calcium compounds in synovial fluid^[1]
3.1 Synovial fluid preparation: Aspirate the synovial fluid into a disposable syringe and transfer the fluid to a clean test tube containing sodium heparin anticoagulant.
3.2 Staining: Aspirate a drop of synovial fluid from the bottom of the test tube, place it on a clean slide, mix it with a drop of 2% Alizarin Red S, and cover it with a coverslip.
3.3 Within three minutes of mixing, observe it under an ordinary light microscope and grade the degree of orange-red staining observed in each high-power field. The area of ??the red area is proportional to the content of calcium compounds.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Alizarin Red S staining of bones in vivo^[3]
(1) Rat treatment: 20% Alizarin Red S (100 mg/kg, single dose) was injected into albino rats and the rats were killed 2 min-8 h after injection.
(2) Skull treatment: The rat skull was cut off and stored in 2% potassium hydroxide solution for several days. All soft tissues were removed, separated and rinsed in tap water, and stored in 80% ethanol. The skull was embedded in unsaturated polyester resin and cut into 50-60u sections in sagittal or frontal planes with a diamond saw.
(3) Microscopic examination: At 80 times magnification, bones emit green autofluorescence and Alizarin Red S emits red fluorescence.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

320.27

C₁₄H₈O₇S

83-61-4

Solid

Yellow to brown

O=S(C(C=C1C2=O)=C(O)C(O)=C1C(C3=C2C=CC=C3)=O)(O)=O

Room temperature in continental US; may vary elsewhere.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. H Paul, et al. Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum. 1983 Feb;26(2):191-200.  [Content Brief]

  [2]. Soomin Lim, et al. Stimulatory Effects of KPR-A148 on Osteoblast Differentiation and Bone Regeneration. Tissue Eng Regen Med. 2019 Jul 17;16(4):405-413.  [Content Brief]

  [3]. H Vilmann, et al. The in vivo staining of bone with alizarin red S. J Anat. 1969 Nov;105(Pt 3):533-45.  [Content Brief]

  [4]. W Querido, et al. Giemsa as a fluorescent dye for mineralizing bone-like nodules in vitro. Biomed Mater. 2012 Feb;7(1):011001.  [Content Brief]

  [5]. Bo Lai, et al. Application of excitation and emission matrix fluorescence (EEM) and UV-vis absorption to monitor the characteristics of Alizarin Red S (ARS) during electro-Fenton degradation process. Chemosphere.2013 Nov;93(11):2805-13.  [Content Brief]