B16-F10R cells invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assays respectively. The initial number of cells cultured was 5 × 105, a sterile 10 µL pipette tip was used to mark a line in the monolayer of each well, the wounds were observed at 0, 24 and 48 h under a microscope (Nikon, Japan). ImageJ software was used to measure the wound areas. Cell free migration area =(cell free area 0 h - cell free area 24h)/cell free area 0 h (Ji et al., 2018 (link)). 5 × 105 cells were placed into the Matrigel-coated invasion chambers (Corning, USA) to incubation, the upper and lower cultures were separated by polycarbonate membranes, and the cells under study were planted in the chamber, after 48 h, the top of Matrigel and invading cells were fixed and stained with 0.1% crystal violet. The microscope (Nikon, Japan) was used to photograph invading cells, and 3 fields were randomly selected and counted each time.
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