Alizarin red S (ARS) staining and quantification

MSCs are believed to accumulate Ca²⁺ and inorganic phosphate during osteogenesis, which serve as nucleating agents for the formation of hydroxyapatite (Ca₁₀(PO₄)6(OH₂)2), the primary inorganic component of bone [19, 20]. Alizarin red S can react with calcium ions to form orange-red complexes that can be directly observed by the naked eye or with a microscope [19]. Thus, Alizarin red S staining can be used to quantify the amount of extracellular mineralized matrix, which is a long-term cumulative effect of osteogenesis [19].

MSCs were first fixed in 4% paraformaldehyde for 30 min and then stained with 1% ARS (pH 4.3) for 15 min at room temperature. To remove nonspecific staining, the stained cells were then washed at least three times with phosphate-buffered saline (PBS). Subsequently, the stained cells were observed under a microscope and photographed. For ARS quantification, 10% cetylpyridinium chloride monohydrate (Sigma-Aldrich) was used to destain the cells for 1 h at room temperature. Thereafter, 200 μL of the liquid was transferred to a 96-well plate, and the spectrophotometric absorbance was measured at 562 nm.