Kindlin-3 recruitment to the plasma membrane precedes integrin high affinity during arrest. (A) K3KO HL-60 cells expressing TagRFP-K3 were labeled with the membrane dye CMDR and rolled at 6 dyn/cm² on coverslips coated with 5 μg/mL P-selectin-Fc and 10 μg/mL ICAM-1-Fc. Then, 100 ng/mL IL-8 was perfused in the presence of 1 μg/mL mAb24-Alexa Fluor 488 (mAb24-AF488). The cell rolling and arrest were observed using qDF imaging. Flow direction was from top to bottom. Merged images show mAb24-AF488 (green) and TagRFP-K3 (red). Scale bar, 5 μm. (B) Integrated fluorescence intensity (IFI, mean fluorescence intensity × area) of kindlin-3 and mAb24-AF488 antibody labeling is depicted as a function of time before and after arrest at 0 seconds. (C) IFI signal after normalization to the membrane (IFI_K3 or mAb24/IFI_CMDR×10 000). (D) Quantification of TagRFP-K3 and mAb24-AF488 binding during cell rolling and arrest in 3 independent experiments. (E-F) 3D distributions and dynamics of high-affinity β₂ and kindlin-3 clusters in neutrophils. Membrane signals were converted to hills (microvilli) and valleys (space between microvilli). Top (E) and side (F) views of the 3D topography of membrane (gray) were overlaid with kindlin-3 (red) and mAb24 (green) clusters. Yellow, colocalization. (F) Horizontal bar represents 5 μm; vertical bar, 100 nm. *P < .05.