Ultrastructure and composition of ENDS. (A) SEM of a manually graded coverslip, CD16-AF647 antibody–labeled human neutrophil, or human ENDS that were centrifuged on the coverslip. Imaged with confocal microscopy and then with SEM. Left scale bar indicates 5 µm, and right scale bar indicates 3 µm. (B) Comparison of ENDS proteome to published proteomes of neutrophil-derived EVs, NETs, neutrophil-derived primary granules (PG), secondary granules (SG), tertiary granules (TG), ficolin granules (FG), and secretory vesicles (SV), and neutrophil plasma membrane (PM). ENDS proteome was also compared with neutrophil lysate (Neut) that was prepared from one of the donor samples that was used to makes ENDS. (C) Gene ontology analysis of ENDS and polymorphonuclear neutrophils proteome. (D) ENDS quantified with confocal microscopy in blood plasma of Ly6G-cre-mTmG mice before and 2 h after i.p. LPS injection. (E) Human ENDS were surface labeled with CD16-AF647 and CD66b-FITC antibodies. Top panel shows overlay of CD16-AF647 (magenta) and CD66b-FITC (green), and bottom panel shows CD66b-FITC alone. CD66b antibody-labeled ENDS, but to a weaker extent compared with CD16 antibody. Scale bar, 10 µm. (F) Human ENDS were surface labeled with CD16-AF647 and antibodies against TS (CD9-PE, CD63-PE, and CD81-PE). Top panel shows overlay of CD16-AF647 (magenta) and labeling for TSs (green). Bottom panel shows TS labeling alone. TSs were present only on some ENDS and in a patchy way. One example of two independent experiments are shown. Scale bar, 10 µm.