Neutrophils release exosomes containing LTB₄ and LTB₄-synthesizing enzymes upon fMLP addition.

<p><b>(A)</b> Differentiated mCherry-5LO cells were plated on fibronectin-coated plates, and time-lapse images were captured before and after a uniform stimulation with 10 nM fMLP. Fluorescent images are representative of six independent experiments. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s010" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s011" target="_blank">S2</a> Movies. <b>(B)</b> Differentiated mCherry-5LO cells were allowed to chemotax under agarose towards an fMLP gradient. Time-lapse images were captured 30 min after the addition of fMLP, 700 μm away from the chemoattractant well. Gradient slope ~ 50 pM/μm (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#sec010" target="_blank">Materials and Methods</a>). Fluorescent images are representative of 20 independent experiments. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s012" target="_blank">S3 Movie</a>. <b>(C)</b> Differentiated CD63-GFP/mCherry-5LO coexpressing cells were allowed to chemotax as described in B. Fluorescent images are representative of eight independent experiments. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s003" target="_blank">S1 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s013" target="_blank">S4 Movie</a>. <b>(D)</b> Negatively stained EM images of pellets obtained from filtration and ultracentrifugation of supernatants of fMLP-stimulated neutrophils (upper panel) and after further purification on a discontinuous iodixanol gradient (lower panel). Vesicle sizes were determined from five vesicles from five fields from three independent experiments. <b>(E)</b> Equal amounts of protein from purified exosomes, crude ultracentrifugation pellets, and total cell lysates (10 μg) were subjected to western analysis using Calnexin, GRP94, CD81, and CD63 specific antibodies. Results are representative of three independent experiments. <b>(F)</b> Detection of CD63 (upper panel) or CD81 (lower panel) on exosomes obtained from fMLP-stimulated or dimethyl sulfoxide (DMSO)-treated neutrophils in a bead-based flow cytometry assay. Relative median fluorescence intensities obtained from three independent experiments are shown as mean ± standard deviation (SD). *** and NS (Not Significant) indicate <i>p</i> < 0.0001 and <i>p</i> > 0.05, respectively, compared to the isotype control. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s004" target="_blank">S2 Fig</a>. <b>(G)</b> Crude ultracentrifugation pellets were loaded on a discontinuous iodixanol gradient, total protein from each fraction precipitated and subjected to western analysis using antibodies for the exosomal marker HSC70 or LTB₄ synthesizing enzymes. 5% of the total input was analyzed. Results are representative of three independent experiments. <b>(H)</b> Representative immunogold EM images of exosomes from stimulated neutrophils stained with CD63 or 5-LO antibodies. <b>(I)</b> Purified exosomes from fMLP-stimulated neutrophils treated with DMSO or MK886 (1 μM, 30 min) were lysed and their LTB₄ content measured using EIA. Results from four independent experiments are shown as mean ± SD. *** and NS indicate <i>p</i> < 0.0001 and <i>p</i> > 0.05, respectively, compared to the LTB₄ content of exosomes purified from vehicle treated nonstimulated cells.