Cdc42 regulates neutrophil polarity via CD11b. (A) Deadhesion. WT and Cdc42^−/− neutrophils were stimulated with fMLP and on slides coated with anti-CD11b to enforce CD11b activation. The stimulus was removed 30 minutes after adhesion, and the remaining adherent fraction was enumerated 10 minutes after stimulus removal at the light microscope (mean ± SD; n = 3 independent experiments, as in Figure 3). (B) WT and Cdc42^−/− neutrophils were stimulated with fMLP and on Fg-coated slides or on slides coated with anti-CD11b to enforce CD11b activation, compared with isotype control. The cells were then fixed and stained with rhodamine-phalloidin (in red) to analyze F-actin structures. The black-and-white pictures represent the phase-contrast images. The images are one x-y view of the z-series analyzed by deconvolution in Volocity. Histograms are number of cells with more than one protrusion (percentage, mean ± SD; n = 3 independent experiments); at least 30 cells per experiment were analyzed. Surface area of F-actin (square microns) and total cell spreading (square microns) are shown as mean ± SD of at least n = 30 cells from at least 2 independent experiments. Arrows point to actin protrusions. Scale bar represents 5 μm. The slides were mounted with Slowfade Gold antifade reagent. Z series of fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63×/1.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software and analyzed by deconvolution with Volocity software. (C) Migration responses of cells plated on anti-CD11b–coated surface was analyzed in a Zigmond chamber in fMLP gradient as in Figure 1. The schema represents the migration trajectory of cells moving up fMLP gradient for 25 minutes. Straightness of migration is indicated as mean ± SEM; n = 70 cells from 3 independent videos. Histogram represents percentage of cells with change in direction arising from inappropriate lateral protrusions (mean ± SD; n = 3 independent videos).