Structure and composition of human ENDS. (A) SEM and confocal image of ENDS formed on P-selectin substrate by human neutrophils surface labeled with CD16-AF647 antibody (magenta). Scale bar, 1 µm. (B) ENDS formed on E-selectin labeled with CD16-AF647 antibody were analyzed with STORM. Image shows a section of ENDS. Dots indicate the localization of the labeling antibody detected with STORM. Certainty of localization ≤20 nm. Scale bar, 100 nm. (C) The frequency histogram shows the distribution of CD16-AF647 labeling antibody across the width of ENDS measured as in B. Data were collected from two independent experiments (black and gray curves). The distance between the vertical red lines indicates mean ENDS diameter determined as the full width at half maximum (FWHM) value. Bin size is 5 nm. (D) To test the presence of organelles in ENDS, neutrophils were labeled for their surface (CD16-AF647 antibody, magenta), cytoplasm (CellTracker Orange CMRA, yellow), ER (ER Tracker Green), mitochondria (Mito Tracker Green), or nucleus/DNA (Hoechst, cyan) and rolled on E-selectin substrate. Only the cytoplasm tracer was detectable in ENDS formed by these neutrophils. Scale bars, 10 µm. Each of the experiments shown on this figure were repeated at least twice.