Image analysis techniques and quantification method application. Time-lapse images were obtained using a Zeiss Axiovert 200 M (2.5× Plan-NEOFLUOR objective, NA 0.075; scale bar = 500 μm) from application of the CWA (A) and CIA (B) methods. Each row of each panel represents one of three cell lines with various levels of aggressiveness (MCF-7, SKOV-3, and MDA-MB-231) captured at 0 h, 24 h, and the difference measured (in pixels) between the areas of those time points. The original, untouched wound for each cell line at 0 h is included in column 1 of each panel. The highlighted red regions (columns 2 and 3) indicate the pseudo-color applied (via ImageJ) to the wounded areas at both 0 h and 24 h, and the blue regions (column 4) indicate the differences (in area) obtained between those time points. Clearly, the Matrigel™ overlay has a considerable effect on wound closure of these cell lines.