Alizarin Red S (sodium)

Alizarin Red S sodium is an anthraquinone derivative dye. When combined with cations such as calcium ions, the functional group of Alizarin Red S sodium can form a coordination bond with the cation through the oxygen atom to show orange-red fluorescence. Alizarin Red S sodium 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 Vitro:Preparation of Alizarin Red S sodium^[1]
(1) Prepare a 2% aqueous solution of Alizarin Red S sodium.
(2) Use a pH meter to add ammonium hydroxide dropwise while stirring to adjust the pH to 4.2.
(3) Filter the solution through a 0.45 μm microfilter and store in an amber bottle.
(4) Refilter the solution through a 0.22 μm microfilter immediately before use.
Alizarin Red S sodium detection of osteoblast differentiation^[2]
(1) Induction of osteoblast differentiation: MC3T3-E1 cells were seeded in four-well plates at 1×10⁵ cells per well and cultured in α-MEM (without ascorbic acid) containing 10% fetal bovine serum. Osteoblast differentiation was induced using osteogenic medium (OSM) containing ascorbic acid (50 μg/mL) and β-glycerophospholipids (10 mM).
(2) Staining: MC3T3-E1 cells were washed with 1×PBS, fixed with 70% ethanol at room temperature for 10 min, rinsed with distilled water, and stained with Alizarin Red S for 10 min to detect calcium deposition in the matrix.
(3) Data analysis: The area of ​​the red zone is proportional to osteoblast differentiation.
Alizarin Red S sodium Screening test for calcium compounds in synovial fluid^[1]
(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.
(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) 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. In Vivo:Alizarin Red S sodium staining of bones in vivo^[3]
(1) Rat treatment: 20% Alizarin Red S sodium (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 sodium emits red fluorescence.