Imagelock tissue culture microplate

A431 cells were transfected with control siRNA, OVOL1 siRNA, or OVOL2 siRNA and were seeded at 1 × 10⁴ cells per well on a 96-well ImageLock tissue culture microplate (4379; Essen Bioscience, Ann Arbor, MI, USA) pre-coated with type I collagen (637-00653, Cellmatrix Type I-A; Nitta Gelatin Inc., Osaka, Japan). Forty-eight hours post-transfection, we scratched the cell monolayers with a wound maker 96 (9600-0012; Essen Bioscience). The wound area of each well was automatically imaged every 2 h in a CO₂ incubator using a live-cell imaging system (IncuCyteHD; Essen Bioscience). We measured the wound area relative to that at 0 h using IncuCyte software (9600-0012, a set of wound markers; Essen Bioscience). We conducted all assays by using 24 wells/condition and repeated them at least three times in separate experiments.

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).

To investigate the effect of NECTIN4 inhibition on cell migration, A431 cells were seeded in a collagen-coated 96-well ImageLock tissue culture microplate (Essen Bioscience, Ann Arbor, MI, USA; 4379) at a density of 2.0 × 10⁴ cells per well, mixed with siRNA–Lipofectamine complexes, and incubated for 48 h. The cell monolayers were then scratched using a wound maker (Essen Bioscience) and the area of the wound in each well was automatically captured every 2 h using an IncuCyte live cell imaging system (Essen Bioscience). The wound area relative to that at 0 h was monitored and recorded using IncuCyte software (Essen Bioscience).