Figure 1

Schematic of Nest Expansion Assay (NEA). Slightly altering our previous Circular Invasion Assay (CIA), the NEA was created by purposely tilting a sterile, flat-bottomed, silicone-tip fitted to a drill press, to leave an ~800 μm circular nest, or island of cells within the wounded area, in order to examine outward growth of cells, which mimics the directional spread of a tumor mass in vivo. Post wounding, Matrigel was laid and allowed to polymerize for 30 min, dishes were incubated at 37°C for indicated times, and nest expansion was calculated by comparing the area of nests at 0 h to the corresponding area at a time point of interest, using Java's ImageJ.