(A) Mock-loaded continuous iodixanol gradients were fractionated, and the density of each fraction determined as described. (B) Nitrogen cavitates of freshly isolated primary human neutrophils were density fractionated on iodixanol gradients, and LTB₄ content from each fraction was extracted and measured by enzyme linked immunoassay (EIA). (C) Total protein from each fraction of iodixanol gradient described in panel B was precipitated using trichloroacetic acid, and western analysis was performed using specific antibodies. Data are representative of six independent experiments.

Representative immunogold EM images depicting the distribution of 5-LO in various cellular compartments in neutrophil chemotaxing towards fMLP. (A) Immunogold image stained with a normal rabbit IgG. (B–E) Immunogold images stained with a 5-LO-specific antibody. (F) Quantification of 5-LO positive staining in MVB, non-MVB vesicles, and mitochondria. (G–H) Progressive magnification of sections (i through iii) of neutrophils migrating towards fMLP showing formation and release of 5-LO-containing MVBs. (I) Fusion of 5-LO-containing MVB with plasma membrane and release of exosomes in cell synapse. Ne: nuclear envelope, Nu: nucleus, MVB: multivesicular body, ILV: intraluminal vesicles, Ex: exosome, PM: plasma membrane, CS: cell synapse.

(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 six independent experiments. Also see and 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 ~ 50 pM/μm (see ). Fluorescent images are representative of 20 independent experiments. Also see . (C) 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 and . (D) 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. (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 three independent experiments. (F) 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 p < 0.0001 and p > 0.05, respectively, compared to the isotype control. Also see . (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. 5% of the total input was analyzed. Results are representative of three independent experiments. (H) Representative immunogold EM images of exosomes from stimulated neutrophils stained with CD63 or 5-LO antibodies. (I) 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 p < 0.0001 and p > 0.05, respectively, compared to the LTB₄ content of exosomes purified from vehicle treated nonstimulated cells.

(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.

(A) Exosomes were purified from differentiated control (NS shRNA), Rab27a shRNA (sh1; sh3), or SMPD2 shRNA (sh2; sh4) KD cells after treatment with fMLP (2 nM, 30 min) and analyzed using a bead-based flow cytometry assay with CD63-FITC, CD81-PE, and CD11b-APC conjugated antibodies. Panels show quantitative analysis from three independent experiments as mean ± SD. Evaluation of exosome flow cytometry was done as described in the legend of . See , for flow cytometry graphs. (B–C) Exosomes were purified from differentiated control and shRNA KD cells 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 in pg/ml/10⁸ cells (B) or pg/ml/μg of exosome protein (C). *** and NS indicate p < 0.0001 and p > 0.05, respectively, compared to corresponding control PLB-985 cells. (D) Differentiated control and shRNA KD cells were stimulated with subsaturating (1 nM) or saturating (1 μM) fMLP for 10 min, and the amount of LTB₄ in the supernatant was assessed by EIA. Results from three independent experiments are shown as mean ± SD. The symbols ** and *** indicate p < 0.001 and p < 0.0001, respectively, compared to corresponding NS shRNA controls. (E) EZ-Taxiscan chemotaxis towards 1 nM of control and KD cell lines. Corresponding migration speeds and CI were calculated from four different experiments and represented as mean ± SD. See legend of for details. Also see . (F) Differentiated NSshRNA, Rab27a, or SMPD2 KD cells or PLB-985 cells overexpressing LTB₄R1 were plated on fibronectin-coated plates for 10 min and uniformly stimulated uniformly with 1 nM fMLP. At specific time points, samples were subjected to western analyses using an antibody against phospho and total myosin light chain II (MLCII). Data are representative of three independent experiments. See , for quantification.

(A) Cell tracks of neutrophils chemotaxing towards a ~50 pM/μm gradient of fMLP under agarose (see ). (i) DMSO-treated cells stained with cytotracker red. (ii) MK886-treated cells stained with cytotracker red. (iii–iv) MK886-treated cells stained with cytotracker red mixed with DMSO-treated cells stained with cytotracker green: (iii) shows the tracks of MK886-treated red cells in the mixture and (iv) shows tracks of DMSO-treated green cells in the mixture. Speed and CI were calculated from the tracks of 40 cells and averaging over six independent movies. The temporal location of cells in the X or Y direction is coded according to the color map shown. Also see . (B) Cell tracks of neutrophils chemotaxing towards fMLP as described in legends of A. In this case, neutrophils were treated with GW4869. Red cells were tracked in panels i, ii, and iii. In panel iv, DMSO-treated cells stained with cytotracker green were tracked. Speed and CI were calculated from the tracks of 40 cells and averaging over six independent movies. Also see and . (C) Graphs depicting the change in the speed of control- (labeled blue) or inhibitor- (labeled red) treated cells as a function of time. The mean average change in the angular deviation (Δα) between two consequent tracks is presented in the bar graph. Data were calculated from the tracks of 40 cells and averaging over six independent movies. *** indicates p < 0.0001 compared to DMSO treated cells.

Tracks, speed, and CI of mixtures of cytotracker-labeled cell lines migrating under-agarose towards a ~50 pM/μm gradient of fMLP. Also see and . (A) Differentiated cells overexpressing the LTB₄R1 (labeled with cytotracker green, shown as blue tracks) in a mixture with NS shRNA cells (labeled with cytotracker red, shown as red tracks) treated with DMSO (left) or CsH (right). Composite graph shows displacement tracks of both types of cells. Speed and CI were calculated from the tracks of 40 cells and averaging over 8 independent movies. (B) Differentiated cells overexpressing the LTB₄R1 (labeled with cytotracker green, shown as blue tracks) treated with CsH in a mixture with NS shRNA cells (labeled with cytotracker red, shown as red tracks) treated with DMSO (left) or MK866 (right). Composite graph shows displacement tracks of both types of cells. Speed and CI were calculated from the tracks of 40 cells and averaging over 4 independent movies. (C) Differentiated cells overexpressing the LTB₄R1 (labeled with cytotracker green, shown as blue tracks) in a mixture with Rab27a sh1 (labeled with cytotracker red, shown as red tracks) or SMPD2 sh2 (right) cells treated with DMSO. Composite graph shows displacement tracks of both types of cells. Speed and CI were calculated from the tracks of 40 cells and averaging over 4 independent movies. The symbols ***, **, *, and NS indicate p < 0.0001, p < 0.001, p < 0.01, and p > 0.05 respectively compared to CsH treated PLB-LTB₄R1 cells.

Cartoon depicting two neutrophils chemotaxing toward an fMLP gradient (green). LTB₄ released from exosomes are shown in blue. See text for details.