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].
In Vivo, 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.
In Vitro, 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.
References:
[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.
[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.
[3]. H Vilmann, et al. The in vivo staining of bone with alizarin red S. J Anat. 1969 Nov;105(Pt 3):533-45.
[4]. W Querido, et al. Giemsa as a fluorescent dye for mineralizing bone-like nodules in vitro. Biomed Mater. 2012 Feb;7(1):011001.
[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.
In Vivo, 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.
In Vitro, 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.
References:
[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.
[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.
[3]. H Vilmann, et al. The in vivo staining of bone with alizarin red S. J Anat. 1969 Nov;105(Pt 3):533-45.
[4]. W Querido, et al. Giemsa as a fluorescent dye for mineralizing bone-like nodules in vitro. Biomed Mater. 2012 Feb;7(1):011001.
[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.