CD11b is critical for polarity of neutrophils during migration. (A) F-actin (rhodamine-phalloidin) analysis of WT, CD11b^−/−, and Cdc42^−/− neutrophils 10 minutes after stimulation with fMLP and on glass. The black-and-white pictures are the phase-contrast images. The images are one x-y view of the z-series analyzed by deconvolution in Volocity. Histogram represents total surface area and area of lamellipodia (mean ± SD of n = 30 cells) and number of cells with more than one protrusion (mean ± SD; 3 independent experiments). Arrows indicate F-actin protrusions. Scale bar represents 5 μm. (B) WT cells were treated with functional anti-CD11b blocking antibody or isotype control for 20 minutes at room temperature and stimulated with fMLP and on fibrinogen-coated slides in the presence of antibody. The cells were analyzed for F-actin structures with rhodamine-phalloidin (mean ± SD; n = 30 from 3 independent experiments). The slides were mounted with Slowfade Gold antifade reagent. Z series of fluorescence images were captured using a Leica DMIRB or 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 of cells analyzed by time lapse video microscopy in a Zigmond chamber in gradient of fMLP on glass, as in Figure 1. Schema of cell trajectory is shown. Straightness of migration is indicated as mean ± SEM. *Results that are significantly different from WT (P < .001). Histogram represents percentage of cells that developed lateral protrusions during the course of migration and changed direction (mean ± SD; n = 3 independent videos). Only cells that had moved more than 20 μm were analyzed. Images were captured at 37°C using a Zeiss Axiovert 200 microscope at 10×/0.3 NA objective, with ORCA-ER C4742-95 camera driven by Openlab software.