Recruitment of kindlin-3 to the plasma membrane during mouse neutrophil arrest. (A) Generation of mice reconstituted with EGFP-K3 and K3KO leukocytes for qDF and intravital imaging. Representative flow cytometric analysis of EGFP-K3 expression (percentage of positive cells indicated) in neutrophils from mouse whole blood (right). (B) Bone marrow cells were flushed from recipient mice reconstituted with EGFP-K3 (green) and labeled with 1 μg/mL anti-Ly6G antibody (Ly6G-AF647, magenta). qDF imaging was performed in mouse neutrophils rolling on mouse P-selectin/ICAM-1 substrate at 6 dyn/cm² in a microfluidics-based flow chamber. Neutrophil arrest was induced by application of 10 ng/mL CXCL1. Flow direction was from top to bottom. Scale bar, 5 μm. Results are representative of 3 independent experiments. (C) Fluorescence (EGFP-K3 and Ly6G-AF647) images of neutrophils at the indicated time points before and after application of CXCL1. Vessel walls are outlined by broken white lines. Horizontal arrow indicates the direction of blood flow. Blue dashed lines indicate positions of arrested neutrophils (white, EGFP-K3⁺Ly6G⁺). Scale bars, 20 μm. (D) The number of adherent neutrophils in mouse cremaster muscle venules of reconstituted mice, before and after injection of 300 ng CXCL1 via the carotid artery catheter. Data are shown as means ± SEM; n = 4 observations in 4 vessels after 4 individual CXCL1 injections. MSCV, murine stem cell virus.