Cdc42 regulates CD11b distribution. (A) WT and Cdc42^−/− neutrophils were stimulated or not stimulated with fMLP and on Fg-coated slides for 10 minutes. The cells were fixed and stained with anti-CD45 (in red) or anti-CD44 (in red) and anti-CD11b (in green). 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. Two-dimensional representation of mean intensity of fluorescence of CD11b and CD45 or CD44 of the region of interest indicated by the box, analyzed in ImageJ. Arrows point to the cell front. Histogram is ratio of mean intensity of fluorescence of CD11b along the sides of the cells to the front normalized to CD45 or CD44 (mean ± SD; n = 55). Scale bar represents 5 μm. (B) The average of fluorescence intensity at the substrate contact surface of CD11b and CD44 was measured along the lateral sides of the cells in at least 5 sections per cell (supplemental data). Histogram shows mean ± SEM of all measurements per cell and of n = 20 cells, representative of 3 independent experiments. The slides were mounted with Slowfade Gold antifade reagent. 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. Scale bar represents 5 μm.