Cell Migration Tracking Protocol

Cells were plated on 35-mm µ-Dish culture plates (81156, ibidi, USA Inc., Madison, WI, USA) that were coated with laminin 332 matrix [57 (link)], at a density of 1 × 10⁴ cells/cm². Images of cells cultured at 37 °C in a humidified atmosphere containing 5% CO₂ were acquired over a 16-h period, using an EVOS Cell Imaging System equipped with an EVOS onstage incubator (Thermo Fisher Scientific), and capturing a phase-contrast image every 10 min. Image sequences were imported into Fiji software [58 (link)] and the Manual Tracking Plug-in was used to track every cell within each frame. Migration tracks were then analysed using the Chemotaxis and Migration Tool software (ibidi). The migration path of each cell was visually outlined, and the accumulated (total) distance, the euclidean distance (straight linear distance from the initial to the final cell position point) and the average speed of each cell were calculated.