Annexin 5 propidium iodide staining solution

For apoptosis analysis, single cells were seeded into the wells of 6-well plates (5 × 10⁵ cells/well) and incubated for 24 h; after which, 1,25(OH)₂D₃ was added and the cells were incubated for an additional 48 h. Next, the cells were harvested, digested to single cells using 0.25% trypsin (Gen-View Scientific, Inc., El Monte, FL, U.S.A.), and prepared for apoptosis detection that was performed after adding 200 µl of Annexin V/Propidium Iodide (PI) staining solution (BD Biosciences). For the EdU proliferation assay, cells in 96-well plates were first treated with 1,25(OH)₂D₃ for 48 h and then treated with 50 μM EdU solution for 2 h (100 μl/well, RiboBio, Guangzhou, Guangdong, China). The cells were then analyzed by flow cytometry as described in the manufacturer’s instructions. All flow cytometric analyses were performed using a BD FACS Calibur™ flow cytometry (BD Biosciences).