Myo1f was required for neutrophil transmigration. (A-B) Myo1f^+/+ and Myo1f^−/− neutrophils were stained with SiR-actin (100 nM) and perfused through rmICAM-1 (12.5 µg/mL) and rmP-selectin (10 µg/mL) coated μ-Slide Membrane ibiPore flow chambers with 1 dyne/cm² shear stress. Time-lapse video microscopy was performed using spinning-disk confocal microscopy. (A) Orthogonal view of representative pseudo-colored time-lapse images demonstrating the localization of neutrophils during the process of transmigration into a fMLP-containing collagen chamber after stimulation for 60 minutes. Scale bar, 10 µm. Color scales, heat map. Triangle indicates orientation of fMLP gradient. Arrow indicates direction of flow. (B) Quantitative analysis of transmigrating Myo1f^+/+ and Myo1f^−/− neutrophils respective to their position in percentage of all neutrophils analyzed (100%). n = 4 independent experiments (with a total of 240 Myo1f^+/+ neutrophils and 220 Myo1f^−/− neutrophils analyzed). Mean ± SEM; ***P < .001; ****P < .0001 (2-way ANOVA, Sidak multiple comparison test). (C-D) Transwell assay without (−) or with CXCL1 either coated with (C) an endothelial monolayer, or (D) uncoated as control. n = 4; mean ± SEM; ****P < .0001.