Cdc42 is critical to maintain front/back polarity of neutrophils during migration. (A) Expression of Cdc42 in WT and Cdc42^−/− neutrophils analyzed by immunoblot. (B) Neutrophil migration using a Boyden chamber in uniform concentration or in a gradient of 1 μM fMLP in which fMLP is placed only in the lower chamber to measure chemokinesis or chemotaxis, respectively. The histogram represents the number of migrated neutrophils per field (mean ± SD, representative experiment in triplicate of 3 independent experiments). (C) Neutrophil migration using transwells coated with fibrinogen in uniform concentration or in a gradient of 10μM fMLP. The histogram represents the total number of migrated neutrophils recovered from the bottom well (mean ± SD, representative experiment in triplicate of 3 independent experiments). (D) Neutrophil migration was examined by time-lapse video microscopy in a gradient of 10μM fMLP and on surface coated with fibrinogen, in a Zigmond chamber. Representative images (1 minute between each frame) of migrating cells, fMLP concentration increases from left to right. Arrows point to inappropriate protrusions. Cell trajectory analysis: the schema represents the migration trajectory of cells moving up fMLP gradient for 25 minutes. Trajectories were tracked with Volocity software and realigned to the same horizontal axis. The black circle represents the starting position. Cdc42-deficient neutrophils exhibited overall displacement toward the fMLP gradient. Speed (sp, μm/min) and straightness (st) of migration are indicated on the right. Data are mean ± SEM; n = 100. The histogram represents the percentage of cells exhibiting change in direction arising from inappropriate lateral protrusions (mean ± SD; n = 3 independent videos); at least 50 cells per video were analyzed. Images were captured at 37°C using a Zeiss Axiovert 200 microscope at 10× objective, NA 0.3, with ORCA-ER C4742-95 camera driven by Openlab software (supplemental Videos). *Results that are significantly different from WT (P < .05).