Individual cell tracking

ImageJ software (MTrackJ) was used to quantify migration directionality and speed. Monolayer boundaries were computed by tracking 10 points on the edge of the wound and computing an average to track the edge. For analysis of region dependence in cell migration during wound healing, we divided the first 15 rows of cells from the free edge into rows of 5 cells and defined them as the leading region, transition region, and trailing region, as illustrated in Supplementary Fig. S2a. The position of a cell was defined by its nuclear centroid. For each region, more than 70 cells in the monolayer were tracked at 10-minute intervals. Directionality was defined as the cosine of the angle between cell trajectory and a line perpendicular to the free edge. A cell migrating directly toward the free surface (i.e. perpendicular to the free edge) would have a cosine equal to 1. Cells migrating directionless and randomly would have an average cosine equal to ~0. Cell migration speed is the accumulated migrated distance divided by time.

To analyze changes cell density in collective cell migration, we quantified cell numbers in six regions of 160 µm wide each, with the initial edge of cell sheets in the middle (Fig.S8a). Then we counted the cell numbers at 0 h, 3 h, and 6 h. Cell density was computed by dividing the cell number in each region by the area.