(A) Exosomes were purified from differentiated mCherry-5LO or CD63-GFP cells pretreated with either DMSO or MK886 (1 μM, 30 min) and subsequently stimulated with 2 nM fMLP. The exosomes were lysed and their LTB4 content measured by EIA. Results from four independent experiments are shown as mean ± SD. The symbols ***, **, *, and NS indicate p < 0.0001, p < 0.001, p < 0.01, and p > 0.05, respectively, compared to corresponding DMSO-treated controls. (B) Detection of CD63 on exosomes purified from differentiated mCherry-5LO or CD63-GFP cells that were pretreated with either DMSO or MK886 (1 μM, 30 min) and subsequently stimulated with 2 nM fMLP in a bead-based flow cytometry assay. Relative median fluorescence intensities obtained from four independent experiments are shown as mean ± SD. NS indicates statistical insignificance (p > 0.05) of the number of exosomes derived from MK886-treated cells compared to DMSO controls. (C) Neutrophil adhesion to fibronectin-coated plates was observed before and 15 min after the addition of 10 μg exosomes derived from mCherry-5LO cells. Differential interference contrast images are representative of three independent experiments. (D) LTB4 (10 nM) or exosomes (50 μg/ml) isolated from mCherry or mCherry-5LO cells was added to the neutrophils for 15 min and pAkt S473 and p44/42 MAPK (Erk1/2) (T202/Y204) were determined by western analysis using specific antibodies. Data are representative of four independent experiments. See for quantitation. (E) EZ-Taxiscan chemotaxis towards fMLP, LTB4, exosomes derived from mCherry-5LO expressing cells, or control buffer. The images show paths of individual cells migrating as circles (from red to blue with increasing time) overlaid onto the final frame. Corresponding migration speeds and chemotaxis index (CI) were calculated from three different experiments and represented as mean ± SD. Also see . (F) Neutrophils were treated with DMSO or LY223982 (10 μM for 30 min) and allowed to migrate towards LTB4 or exosomes derived from mCherry-5LO expressing cells. Corresponding migration speeds and CI are calculated from 3 different experiments and represented as mean ± SD. Also see and and Movies. (G) Exosomes derived from mCherry, mCherry-5LO, or CD63-GFP expressing cells were divided into two parts. One part was used to measure LTB4 levels to be added exogenously to neutrophils (control or LY223982 treated). The other part was added to neutrophils and pAkt (S473) levels were assessed after 15 min incubation. Data are representative of three independent experiments. See for quantification.