Fig 3.

Motility characteristics of the cell lines. Vero, HaCaT, or RPE cell monolayers were plated in a two-chamber system with a removable gasket. As described in Materials and Methods, the subsequent gap filling assay was analyzed by time-lapse microscopy after removal of the gasket. (a) Individual snapshots at different times (0 to 18 h) after start of the assay showing gap filling by each of the cell lines. The frontline boundary of moving cells is outlined (black) on one boundary. Regions termed A within the denser internal area and B at the cell boundary are indicated by brackets. Boxes within the 18-h time point indicate typical AOI used to calculate cell density as described in the text. (b) Zoomed image (12-h time point) of the boundaries showing the distinct morphology of the migrating cells at the front. (c) Three independent AOI (black squares) within the defined regions A (original plated area) and B (just behind boundary) were quantified. The average cell density of an AOI in region A was standardized as 100% and then compared to that in the average AOI from just at the boundary. (d) Quantification of migration for 10 independent cells within area A for each cell line. Each cell was identified at the start of the time lapse, tracked (one point/5 min) for 18 h, and positioned from the origin plotted against time. The scales are identical for all cells.