Failure of plasma membrane breakdown and extracellular DNA release by eosinophils. To induce ET formation, eosinophils and neutrophils were stimulated with (A-B, D-G) PMA (100 ng/mL), (C) MSU crystals (200 μg/mL), or opsonized C. albicans for 4 hours (unless otherwise indicated) at 37°C. (A-C) Images were acquired using a Leica SP8 confocal microscope. (A) NETosis dynamics upon PMA stimulation were assessed by live cell imaging using the permeable DNA dye DRAQ5 (cyan) and the impermeable DNA dye Sytox Green (magenta). Asterisks indicate an eosinophil. Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 1. Results are representative of 3 independent experiments. (B) Dynamics of eosinophils upon PMA stimulation. Scale bar = 20 μm. Refer to supplemental Video 1. Results are representative of 2 independent experiments. (C) Dynamics of eosinophils and neutrophils upon exposure to MSU crystals (upper panel) or C. albicans (lower panel). Scale bar = 100 μm. Refer to supplemental Videos 2-3. Results are representative of 2 independent experiments. (D-G) Images were acquired using the ZEISS LSM 980 Airyscan 2 confocal microscope, original magnification ×400. (D-E) Dynamics of eosinophils upon PMA stimulation were assessed by live cell imaging using the plasma membrane dye CellMask Orange (orange), permeable DNA dye DRAQ5 (magenta), and the impermeable DNA dye Sytox Green (green). Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 4. (E) Stills of supplemental Video 5, with a 360º zoom around an eosinophil illustrating that the plasma membrane of the eosinophils does not breakdown and DNA remains inside of the cell. Scale bar = 5 μm. (F-G) NETosis dynamics upon PMA stimulation. Scale bar = 20 μm. Refer to supplemental Video 6. (D) Stills of supplemental Video 7, with a 360º zoom around a neutrophil illustrating that the plasma membrane of the neutrophil breaks down, resulting in extracellular release of DNA and thus NETosis. Scale bar = 5 μm.