A Femtojet Micromanipulator 5171 (Eppendorf-Brinkman Instruments) and a pump, (model Femtojet; Eppendorf) were used to control the position of the micropipette and the pressure required for the chemoattractant flow. U73122 was used to inhibit PLC, and U73343 was used as a control. To induce the formation of protrusion, a micropipette was filled with 25 nM EGF and was placed ∼5 μm from the edge of a quiescent cell, and a pressure of 16 hPa was exerted to induce flow. Time-lapse series were taken using 20× NA 1.4 infinity-corrected optics on a microscope and analyzed in ImageJ. Protrusion is measured along three lines emanating from the cell centroid: (1) the front protrusion is measured along the axis formed by the centroid and the tip of the pipette; (2) the side protrusion is measured along the perpendicular to the latter; and (3) the back protrusion is measured along the axis forming 180° with the front axis. All measurements are standardized over the corresponding distance between the centroid and the cell periphery along the corresponding axis before the introduction of the pipette.