Wound heal assay

The effect of dioscin on cell migration was assessed via a wound healing assay (7). A375 cells and B16F10 cells were seeded at a density of 1,000,000 cells/well and grown to 100% confluence in 6-well plates. The confluent cell monolayer was scratched with a sterile 10 µl pipette tip across the center of each well in order to produce a clean, straight wound area. Cells were subsequently washed twice with PBS in order to remove the detached cells, and incubated with dioscin (0.00, 0.25 and 0.50 µM) in serum-free DMEM medium at 37°C in a humidified atmosphere of 5% CO₂ for 12 h. Cell migration was captured at the 0 and 12 h time points using a digital camera installed on a Leica DM3000 inverted fluorescence microscope (Leica Microsystems Ltd.) with a ×5 objective lens under the bright field mode. A total of five images were captured for each well. Migration rate was calculated using the following equation: (A-B)/A ×100%, where A represents the width of wound at 0 h and B represents the width of wound at 12 h.