WASP is crucial for explosive actin polymerization during pseudopod formation in neutrophils. (A) Spinning-disk confocal images of polymerized actin of control and WASP-KD HL-60 cells after stimulation for the indicated time with chemoattractant (20 nM fMLP) stained with fluorescent phalloidin. (B) FACS quantification of actin polymerization in control (gray circles) and WASP-KD HL-60 cells (green triangles) after stimulation for the indicated time with chemoattractant stained with fluorescent phalloidin. 10,000 cells were counted for each sample, and means were normalized to time 0 for control cells within each experiment to control for detector variability. AU, arbitrary unit. (C) Example spinning-disk confocal images of pseudopod-forming WASP-KD and control cells with polymerized actin stained with phalloidin (red) and DNA stained with DAPI (blue). (D) Quantification of phalloidin staining shown in C. Only cells with pseudopods were analyzed. Mean pixel values for z projections of image stacks was measured for each cell, and then the mean background pixel value was subtracted. Means and SD (bars) from three biological replicates (dots) are shown, with >150 cells total; the p-value was obtained from a one-tailed paired t test.