hMSCs were cultured in osteogenic medium consisting of MEM-α medium, 10% (v/v) FBS, 0.5% (v/v) penicillin-streptomycin, and human osteogenic supplement (R&D Systems, Minneapolis, MN) when the cells were 80%–90% confluent. Osteogenic induction was achieved by maintaining the cells in the osteogenic medium for two or three weeks with medium change every 3–4 days.
ARS staining was used to visualize calcium deposits in cell culture, as described earlier (Rong et al., 2022 (link)). Briefly, cells were washed twice with 1 × PBS and fixed with 10% neutral buffered formalin (Sigma) for 5 min at room temperature. After washing with deionized water, the cells were stained with 1% ARS solution (GFS Chemicals, Columbus, OH, USA) for 5 min and rinsed with water to remove the excessive dye. The calcium deposits were observed under a microscope and imaged with an Olympus Q-Color3™ imaging system.
To quantify calcium deposition in osteogenic differentiated hMSCs, the cells were washed twice with 1 × DPBS followed by calcium collection with 250 μl/well (24-well plate) of 0.6 N HCl solution at 4°C for 2 days. The calcium concentration in the solution was then quantitated by a QuantiChrom™ Calcium Assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s protocol.
ARS staining was used to visualize calcium deposits in cell culture, as described earlier (Rong et al., 2022 (link)). Briefly, cells were washed twice with 1 × PBS and fixed with 10% neutral buffered formalin (Sigma) for 5 min at room temperature. After washing with deionized water, the cells were stained with 1% ARS solution (GFS Chemicals, Columbus, OH, USA) for 5 min and rinsed with water to remove the excessive dye. The calcium deposits were observed under a microscope and imaged with an Olympus Q-Color3™ imaging system.
To quantify calcium deposition in osteogenic differentiated hMSCs, the cells were washed twice with 1 × DPBS followed by calcium collection with 250 μl/well (24-well plate) of 0.6 N HCl solution at 4°C for 2 days. The calcium concentration in the solution was then quantitated by a QuantiChrom™ Calcium Assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s protocol.
