Cdc42 regulates CD11b functioning. (A) Neutrophils were stimulated or not stimulated with fMLP in suspension at 37°C. The reaction was stopped by adding paraformaldehyde, and the cells were stained for CD11b. The arrows indicate small clusters and patches of CD11b in WT cells. CD11b clusters were analyzed in ImageJ software. A 3-dimensional representation of fluorescence intensity, which was generated in ImageJ, is shown. Histogram represents the number of CD11b clusters per cell (mean ± SD; n = 40 cells from 3 independent experiments). Scale bar represents 5 μm. The slides were mounted with Slowfade Gold antifade reagent. Fluorescence images were captured at room temperature using a Leica DMIRB fluorescence microscope at 63× objective, NA 1.3, with ORCA-ER C4742-95 camera driven and analyzed with Openlab software. (B) Adhesion and deadhesion of neutrophils. Neutrophils, stimulated with fMLP in the presence of Ca²⁺ and Mg²⁺, were allowed to adhere to fibrinogen at the indicated time. The nonadherent fraction was removed. The wells were carefully washed with phosphate-buffered saline, and the adherent fraction was immediately enumerated at the light microscope. Deadhesion: 30 minutes after adhesion to fibrinogen, the nonadherent fraction was removed and fMLP was replaced with phosphate-buffered saline without Ca²⁺ and Mg²⁺. The remaining adherent fraction was enumerated at the light microscope 10 minutes after fMLP removal. The histograms represent the number of adherent cells per field (mean ± SD; n = 3 independent experiments).