EVs mediate the autocrine/paracrine action of LTB₄ during neutrophil arrest and extravasation response. (A and B) WT neutrophils labeled with CellTracker Green CMFDA (green) were pretreated with vehicle (DMSO; i) or 5 µM MK886 (ii), 2 µM Nexinhib20 (iii), 20 µM GW4689 (iv), or 40 µM Exo1 (v) for 30 min and coinjected into Alox5^−/− mice with inflamed footpads and Alox5^−/− neutrophils labeled with CellTracker Red CMTPX (red). Maximum-intensity projections of Z-stacks acquired by 2P-IVM (A). See Video 8. Blood vessels are shown in blue, and dashed orange lines indicate vessel boundaries. Orange arrowheads point to extravasated neutrophils. Scale bars = 40 µm. Analysis of the percentage of neutrophils displaying rolling, arrest, and extravasation in the above-mentioned conditions (B). Data are plotted as mean ± SEM from n = 3 mice per group, and each dot represents the result from an individual animal. Two-way ANOVA using Dunnett’s multiple comparisons test was performed to determine statistical significance. (C and D) WT neutrophils were labeled with CellTracker Green CMFDA (green) and fluorescently conjugated M18/2 antibody (red) before their adoptive transfer into Alox5^−/− mice with inflamed footpads and imaged by ISMic. A representative maximum-intensity projection of a Z-stack with blood vessel in cyan (boundary marked with magenta dotted line); blue arrowheads indicate the EVs that rolled and tethered to the inflamed endothelial surface facing the vascular lumen (VL), and PS indicates the perivascular space (C). Scale bar = 5 µm. See Video 9. Quantification of the size of EVs detected using ISMic analysis (D). Each dot represents the value of a single EV, and a total of 60 EVs were analyzed from n = 3 independent experiments. (E and F) Neutrophils purified from WT (E and F) or Alox5^−/− (F) or Itgb2^−/− (F) mice were labeled with CellTracker Green CMPTX and fluorescently conjugated M18/2 antibody (red), adoptively transferred into Alox5^−/− mice with inflamed footpads, and imaged by ISMic. Additionally, WT neutrophils were pretreated with either vehicle (DMSO) or 2 µM Nexinhib20 or 20 µM GW4689 or 10 µM nBleb for 30 min before their adoptive transfer into the recipient. The number of EVs detected was normalized to the number of detected neutrophils for each condition, as described in the Materials and methods section. Each dot represents the result from an individual animal experiment. Data are plotted as mean ± SEM. One-way ANOVA using Dunnett’s multiple comparisons test was performed to determine statistical significance, and no significant difference was observed in F. (G) Neutrophils purified from WT mice pretreated with either vehicle (DMSO) or 2 µM Nexinhib20 for 30 min, labeled with CellTracker Green CMPTX and fluorescently conjugated M18/2 antibody (red), adoptively transferred into infected Alox5^−/− mice with inflamed footpads, and imaged by ISMic. The peripheral Itgb2 redistribution index was determined as described in the Materials and methods section. Each dot represents the value for an individual cell, with values for WT vehicle control the same as in Fig. 3 F and n = 54 cells for Nexinhib20, from n = 3 mice for each group. An unpaired t test with Welch’s correction was used to determine statistical significance. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.