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.