ATF3^−/−neutrophils exhibit a profound defect in directional motion. (A) Representative images (1 minute between each frame) of migrating (upper) WT or (lower) ATF3^−/− neutrophils in a gradient of fMLP on uncoated surfaces in a Zigmond chamber. fMLP concentration increases from left to right. Images were captured every 5 seconds for 20 minutes at 37°C. Original magnification, ×100. The number of neutrophils able to move productively (20 μm from their starting position) is quantified at right. Images are representative of, and graph is pooled from, 4 independent experiments; N = 6 WT and 9 ATF3^−/− independent acquisitions/experiment, and 20 to 100 neutrophils were evaluated/acquisition. Images were captured using a Zeiss Axiovert 200 microscope at 10×/0.3 NA objective, equipped with an ORCA-ER C4742-95 camera driven by Openlab (version 5.5.0) software. (B) Paths of migrating (left) WT and (right) ATF3^−/− neutrophils were traced using ImageJ software. The schema represent cells moving in fMLP gradient over 20 minutes. The total distance traveled from the origin (net translocation) is quantified at right. Schema are representative of, and graph is pooled from, 3 independent experiments; N = 4 WT and 9 ATF3^−/− independent acquisitions/experiment. Statistics are unpaired 2-tailed Student t test. Data are mean ± SEM. **P < .01, ***P < .001.