MPO-mediated PMN motility is highly directed and independent of cytoskeletal rearrangements. (A) Plots of PMN locomotion in microslides are depicted, red lines represent tracks of cells moving toward the up-gradient segment. Cell motility was followed under an Olympus CK2 inverted microscope with a mounted CCD camera (Retiga 1300, QImaging). Time-lapse microscopy was performed with iVision version 4.0 (Biovision) software and tracks created with ImageJ. (B) The mean accumulated distance of plotted tracks was calculated (n = 3-4 plots including 20 tracks, respectively, one-way ANOVA P < .002). (C) The x-forward–migration index (x-displacement × accumulated distance⁻¹) of plotted tracks (n = 3-4 plots including 20 tracks) represents the extent of vectored PMN movement (one-way ANOVA P < .01). (D) PMN administered to microslides in HSA-, MPO-, and IL-8–gradients were visualized with relief contrast microscopy (magnification ×400, digital interference contrast (DIC) microscopy, Olympus CKX31). (E) Motility toward MPO or IL-8 after preincubation of PMN with blebbistatin (dark gray), LY294002 (white), or cytochalasin D (light gray) was tested (n = 3-4, one-way ANOVA P < .0001). (F) Intracellular f-actin content of PMN after exposure to HSA, MPO, or IL-8 for indicated times was assessed by flow cytometry (n = 3-4). (G) Chemotaxis experiments in microslides were performed after preincubation of PMN with sodium azide and 2-deoxyglucose (white bars) to deplete energy metabolism. (n = 3-6, one-way ANOVA P < .0001). In this case, n denotes number of independent experiments, number of donors of PMN ≥ 3. Bars represent means; error bars, SEM. *P < .05, **P < .01, ***P < .001.