WASp^−/− neutrophils have defective chemotaxis and exhibit loss of polarity. (A) WT and WASp^−/− neutrophil migration by time-lapse video microscopy in a gradient of fMLP and on surface coated with Fg, in a Zigmond chamber. Representative images (1 minute between each frame) of migrating cells, fMLP concentration increases from left to right. Images were captured at 37°C using a Zeiss Axiovert 200 microscope at 10× objective N/A0.3 with ORCA-ER C4742-95 camera driven by Openlab Version 5.5.0 software. (B) Cell trajectory analysis; the schema represents the migration trajectory of cells moving up fMLP gradient for 20 minutes. Trajectories were tracked with ImageJ Version 1.43J software. Speed (sp; μm/min) and straightness (st) of migration are indicated at the bottom (mean ± SD; n = 60; *P < .01; 3 independent experiments). (C) Histogram represents the percentage of cells with changes in direction arising from inappropriate lateral protrusions as seen during time-lapse video microscopy. Data are from 80 cells (mean ± SD; 3 independent videos; *P = .00013). (D) Immunofluorescence analysis of F-actin on stimulation with fMLP and Fg, F-actin was labeled with Rhodamine Phalloidin and slides were mounted in SlowFade Gold Antifade. Scale bar, 10 μm. Histogram is percent of cells with multiple protrusions. (mean ± SD; 3 independent experiments; **P < .01). Fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63× objective N/A1.3 with ORCA-ER C4742-95 camera driven by Openlab Version 5.5.0 software.