Wound-Healing Assay and Transwell Migration Assay

The migratory ability of LoVo and Hct116 cells was evaluated by wound-healing and transwell migration assays. Briefly, cells were incubated in a 6-well plate, and at least five horizontal lines were drawn at the back of the plate. The cells were then placed in each well and cultured overnight. Straight scratches were then made vertical to the line drawn on the back of the plate, and the wells were washed three times with PBS to remove excess cells. Cells were cultured in a serum-free culture medium at 37°C with 5% CO₂ and observed at 0 h and 22 h. Thereafter, cell culture inserts (8 µm; Corning Inc.; 24-well plate) were used in the transwell migration assay. Cells (5 × 10⁴) in 200 µL medium (supplemented with 1% FBS) were seeded in the upper chamber, and 600 µL medium (supplemented with 20% FBS) was added to the lower chamber. After incubation for 24 h, the cell culture inserts were fixed with absolute methanol for 30 min and then stained with 0.1% crystal violet for 30 min.