Alizarin Red S sodium (ARS sodium) is an anthraquinone dye widely used to evaluate calcium deposits in cell culture[1].

Solution preparation
Preparation of mother solution: Prepare a 2% Alizarin Red S sodium aqueous solution.
Preparation of working solution: Use ammonium hydroxide to adjust the pH to 4.2, filter the solution with a 0.45 μm microfilter and store it in an amber bottle and re-filter the solution with a 0.22 μm microfilter before use.

I. Detection of osteoblast differentiation by Alizarin Red S sodium
Operation steps
1. Induce osteoblast differentiation: MC3T3-E1 cells were seeded on 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 with 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 area is proportional to osteoblast differentiation.
2. Screening test of calcium compounds in synovial fluid by Alizarin Red S sodium
1. Synovial fluid preparation: Aspirate the synovial fluid into a disposable syringe, and then 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;
c3 Within three minutes after mixing, observe it under an ordinary optical microscope, and grade the degree of orange-red staining density observed in each high-power field of view. The area of ​​the red area is proportional to the content of calcium compounds.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 41.67 mg/mL (121.75 mM), Sonication is recommended.

H2O: 10 mg/mL (29.22 mM), Sonication is recommended.