Q color3 imaging system

Osteogenic differentiation was induced by maintaining the cells in osteogenic medium consisting of DMEM-LG, 10% FBS, 50 μg/ml ascorbate-2 phosphate, 0.01 μM dexamethasone and 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) in 37°C, and 5% CO₂ for 2-3 weeks. The osteogenic medium was changed every 4 days during the incubation. Osteogenic differentiation of rDPSCs was assessed with ARS staining and QuantiChrom™ Calcium Assay Kit (BioAssay Systems, Hayward, CA, USA) with the following methods.
For ARS staining to reveal matrix mineralization/calcium deposition, the cells were washed twice with 1 × PBS and fixed with 10% formalin solution (Sigma) for 5 min at room temperature. The fixed cells were then stained with 1% Alizarin Red S solution (GFS Chemicals) for 5 min at room temperature. After rinsing with ddH₂O to remove the unbound dye, the mineralization/calcium staining was observed under a microscope (Axiovert 25, Zeiss) and photographed with Olympus Q-Color3™ imaging system.
For calcium quantification using QuantiChrom™ Calcium Assay, the cells were washed with 1x DPBS buffer, and then equal volume (250 μl) of 0.6 N HCl was added to each well for overnight incubation at 4°C to collect the calcium [Lee et al., 2014 ]. The calcium content in the HCl solution was assayed following the manufacturer’s instructions.

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.