Incucyte software package

Cell mobility was investigated using the IncuCyte^™ Imaging System (Essen BioScience, Ann Arbor, MI, US). Cells at a density of 50,000 cells/100 μl/well were seeded in an Essen Imagelock 96-well plate and incubated until confluence in a standard CO₂ incubator at 37°C for 24 hours. Uniform wounds were made in each well with the 96-well WoundMaker (Essen BioScience). The medium was removed, and the cells were washed twice with PBS to remove floating cells and debris. The 96-well plate was then inserted into the IncuCyte FLR platform (which includes an automated microscope), and the cells were incubated with 100 μl of medium in the presence or absence of mitomycin C (10 μg/ml) to prevent cell proliferation. Photographs of the same area of the wound were automatically taken within the CO₂ incubator using IncuCyte zoom software (Essen BioScience) at intervals of 3 hours, and the data were analyzed using the IncuCyte software package (Essen BioScience). Cell migration is expressed as the percentage of the gap relative to the total area of the cell-free region immediately after the wound was made. For each well, three randomly selected images were acquired, all experiments were carried out in triplicate, and the results were averaged.