Coro1A controls affinity regulation and clustering of LFA-1 critical for PMN adhesion under flow. In vitro flow chamber assays were performed with dHL-60-Coro1A-EGFP cells by using spinning-disk confocal microscopy. (A) Kinetics of induction of β₂ integrin high-affinity conformation were analyzed by binding of mAb24 antibody. Single-cell velocity and mAb24 intensity values were measured at indicated time points. Data of 3 representative cells from 3 independent experiments are shown. Mean ± standard deviation. (B) Representative pseudocolored snapshots demonstrating the subcellular localization of high-affinity β₂ integrins (mAb24), Coro1A, F-actin, and colocalization of all 3 molecules (white) during rolling and adhesion in single-cell analysis. Arrow indicates direction of flow. (C) Scatter plots of high-affinity LFA-1, Coro1A, and F-actin intensity profiles in single-cell analysis. Colocalization was monitored by calculating the Pearson’s correlation coefficient (P_r) at indicated time points. (D) Intensity profiles of colocalized Coro1A/mAb24, mAb24/F-actin, and Coro1A/F-actin complexes during rolling and adhesion. Data of 3 representative cells from 3 independent experiments are shown. Mean ± standard deviation. (E) Representative pseudocolored overlay of single time points and intensity profiles demonstrating LFA-1 clustering using the Alexa Fluor 594-conjugated anti-CD11a antibody (clone 2D7) and F-actin accumulation in Coro1A^+/+ and Coro1A^−/− PMNs during rolling and adhesion. Arrow indicates direction of flow. A representative PMN from 3 independent experiments with a total of 33 Coro1A^+/+ and 34 Coro1A^−/− PMNs is shown. (F) Quantitative analysis of rolling distance until the onset of adhesion in Coro1A^+/+ and Coro1A^−/− PMNs (n = 3 with a total of 137 Coro1A^+/+ and 24 Coro1A^−/− PMNs). Mean ± standard deviation. Scale bars, 10 µm. Color scales, heat maps. *P < .05.