Figure 5.

EETs are structurally stable and induce platelet adhesion. (A, upper panels) Spontaneous spreading of EETs and NETs. Isolated eosinophils (Eo) and neutrophils (Neut) were stimulated with PMA to induce ET formation. Extracellular DNA was visualized using the cell-impermeable fluorescent DNA dye SYTOX in static culture conditions, and images were obtained using a fluorescence inverted microscope (A, lower panels). Morphological differences between EETs and NETs were determined by SEM. EETs showed bold and condensed chromatin threads. Cell-free eosinophil granules (asterisk) were also observed. (B) SYTOX area per cell was measured using ImageJ software. Results were expressed as mean ± standard deviation (SD). n = 3, ***P < 0.001, Student’s t-test. (C) DNase1 was added to EETs and NETs and dsDNA was quantified using Picogreen intensity. The amount of dsDNA (DNase1/vehicle control x 100) at 0 minutes was considered as baseline. Results were expressed as mean ± SD. n = 3, *P < 0.05, Student’s t-test. (D) EETosis cells were treated with or without DNase followed by incubation with fluorescence-labeled platelet suspension. Adhered platelets per field were counted. Results were expressed as mean ± SD. n = 3, **P < 0.01. (E) SEM image indicated abundant platelets (yellow -colored) adhered to EETs (blue -colored). Platelets were aggregated and formed filopods. Scale bar; 10 µm.