Confocal micorscopy analysis. Glass coverslips were coated by incubating in PBS containing 10 μg/ml of collagen I overnight at 4°C. Circular wounds were made in the DLD-1 cell layer as previously described in Materials and Methods. Cells were further incubated for 5 hr at 37°C in the presence or absence of Matrigel™ overlay and fixed by 3.7% formaldehyde. Cortactin (green; lamellipodial marker) and actin (red; cytoskeletal marker) were visualized by immunofluorescence staining using anti-cortactin antibody 4F11 and Alexafluor 568-phalloidin. Images were obtained with a Zeiss LSM-510 laser scanning confocal microscope equipped with a Plan-NEOFLUAR 40×/1.3 Oil DIC lens (scale bar = 10 μm). Lateral Z-stack images (captured from areas indicated by green, horizontal and red, vertical lines) indicate that in both assays, the cells remain at the plane level of the substrate (dish). In the CIA, cells seem to move along the interface of the dish and Matrigel™ overlay, rather than upwards into the gel. However, slight morphologic changes are observed between CWA and CIA methods at the same time point, indicating that Matrigel™ is providing some form of constraint on cells.