Cell Migration Assay with Wound Healing

Cell migration assay was performed as described in a previous report with slight modifications ^{78 (link)}. Cells were seeded into a 96-well ImageLock tissue culture microplate (1.5 × 10⁴ cells/well; Essen Bioscience, Ann Arbor, MI) pre-coated with type I collagen (Nitta Gelatin Inc., Osaka, Japan) and incubated at 37 °C in 5% CO₂. Twenty-four hours after the incubation, cells were transfected with siRNAs as described above. At 48 h, cell monolayers were scratched with a wound-maker (Essen Bioscience) and images of the scratched area were automatically captured every 2 h for a total of 24 h in the IncuCyte Cell Imaging System (Essen Bioscience). The wound area relative to that at 0 h was calculated by IncuCyte software (Essen Bioscience).