Kindlin-3 is essential for high-affinity integrin activation and HL-60 cell arrest. (A) A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)–based gene-editing tool was used to delete kindlin-3 from HL-60 cells. Western blot detection of kindlin-3 was performed in WT or K3KO HL-60 cells. (B) Differentiated HL-60 cells deficient in kindlin-3 or integrin β₂ (β₂KO) were incubated with 100 ng/mL IL-8 in the presence of the fluorescently labeled, conformation-specific reporters mAb24 and KIM127. Real-time flow cytometry (left) was used to measure the binding of mAb24 and KIM127. Data are the change in median fluorescence intensity (MFI; right). Means ± SEM (n = 5, 5, and 3 individual experiments in WT, K3KO, and β2KO cells, respectively). (C) Fluorescent images of K3KO HL-60 cells expressing kindlin-3 fusion proteins (EGFP-K3 or TagRFP-K3). EGFP-K3 and TagRFP-K3 HL-60 were excited at 488 nm (top) and 561 nm (bottom), respectively. Scale bars, 10 µm. (D) WT, K3KO, or EGFP-K3 HL-60 cells rolled on P-selectin-Fc/ICAM-1-Fc substrate and were perfused with IL-8 (100 ng/mL) in a microfluidics-based cell arrest assay. Arrested cells per field were counted. (E) Integrin activation was measured by flow cytometry in WT and K3KO-, K3-EGFP–, and EGFP-K3–reconstituted HL-60 cells (n = 3, 3, 4, and 5, respectively). **P < .01; ***P < .001; ****P < .0001; n.s., not significant.