ENDS degrade and release S100A8–S100A9 complex. ENDS were produced from human neutrophils on an E-selectin–coated substrate as shown in Fig. 1. (A) ENDS were surface labeled with CD16-AF647 antibody and imaged over 4 h. During that period, ENDS contracted as indicated by their decreasing size (area; black line) and increasing sphericity (red line). Values represent average ± SEM of three independent experiments. (B) Human ENDS were imaged over 4 h in the presence of fluorescently labeled annexin-5 (Ann5) in the incubation buffer. The red outlines indicate the ENDS contours determined based on surface labeling with CD16-AF647 antibody. Black pixels show the thresholded annexin-5 signal. After 4-h incubation, ∼50% of ENDS were annexin-5 positive. Average ± SEM of three independent experiments. (C) Fresh (0-h-old) and 7-h-old human ENDS were loaded with Fluo4-AM to measure intraluminal free Ca²⁺ before and after exposure to ionomycin (Iono). The red outlines indicate the ENDS contours indicated by CD16-AF647 surface labeling. Black pixels indicate the thresholded Fluo4 signal. Values represent average ± SEM of seven independent experiments. Statistical analysis was done using a paired t test; ****, P < 0.0001. (D) S100A8–S100A9 complex concentrations were measured in supernatants of ENDS produced with human neutrophils in a flow chamber. ENDS were incubated in HBSS buffer for 1, 7, or 24 h. Statistical analysis was performed using a ratio paired t test; **, P = 0.0096. Scale bars, 10 µm.