Fig 2. Neutrophils release exosomes containing LTB₄ and LTB₄ synthesizing enzymes upon fMLP addition.

(A) 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 6 independent experiments. Also see S1 and S2 Movies. (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 approximately 50 pM/μm (see Materials and methods). Fluorescent images are representative of 20 independent experiments. Also see S3 Movie. (C) Differentiated CD63-GFP/mCherry-5LO co-expressing cells were allowed to chemotax as described in B. Fluorescent images are representative of 8 independent experiments. Also see S1 Fig and S4 Movie. (D) Upper panel: Representative negatively stained EM image of vesicles after purification on a discontinuous iodixanol gradient. Middle panel: Plot depicting the vesicle sizes determined from 600 vesicles from 3 independent experiments (plotted as mean +/− SEM). Bottom panel: Representative nanoparticle tracking analysis of 3 independent experiments (plotted as mean +/− SEM of 5 technical replicates). (E) 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 3 independent experiments. (F) Detection of CD63 (upper panel) or CD81 (lower panel) on exosomes obtained from fMLP-stimulated or DMSO-treated neutrophils in a bead-based flow cytometry assay. Relative median fluorescence intensities obtained from 3 independent experiments are shown as mean ± SD. *** and NS indicate p < 0.0001 and p > 0.05, respectively, compared to the isotype control. Also see S2 Fig. (G) 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. Approximately 5% of the total input was analyzed. Results are representative of 3 independent experiments. (H) 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 4 independent experiments are shown as mean ± SD. *** and NS indicate p < 0.0001 and p > 0.05, respectively, compared to the LTB₄ content of exosomes purified from vehicle treated nonstimulated cells. Raw data for panels F and H can be found in the Supporting information section S1 Data file. Uncropped blots for panels E and G can be found in the S1 Raw images file. CD63-GFP, GFP-tagged CD63; EM, electron microscopy; EV, extracellular vesicle; FLAP, 5-LO activating protein; fMLP, N-formylMethionyl-Leucyl-Phenylalanine; LTA₄H, LTA₄ hydrolase; LTB₄, leukotriene B₄; mCherry-5LO, mCherry-tagged 5-LO; MFI, mean fluorescence intensity; NS, not significant; 5-LO, 5-lipoxygenase.