Characteristics of EETosis-induced intact eosinophil granule release. (A) Several cell-free granules were released by budding from PMs of eosinophils undergoing ETosis. Eosinophils were studied with time-lapse, phase-contrast imaging every 3 seconds following 1 mg/mL immobilized IgG or 2 μM A23187 stimulation. Images were captured from supplemental Video 1 and supplemental Video 2 (Nikon Eclipse TE300, 100× PlanApo objective with a numerical aperture of 1.40, equipped with a Qimaging Retiga EXi cooled digital camera in conjunction with iVision image analysis software). Eosinophils that initially showed a typical bilobed nucleus then demonstrated that peripheral small PM protrusions developed. Arrowheads show releasing/released granules. After nuclear membrane disintegration to release DNA nets within the cytoplasm, eosinophils gradually turned into a rounded shape, and granule movement slowed with deflected intracellular distribution of the granules. Several extracellular granules remained attached to the culture plate. Data representative of >3 experiments of independent donors with similar results are shown. (B) EETosis-elicited production of subcellular structures (⩽4 μm) was inhibited by DPI. Eosinophils were stimulated with A23187 (2 μM) in the presence of DPI in 0.1% BSA containing RPMI medium for 60 minutes. Subcellular structures in culture medium were counted as described in “Materials and methods” and supplemental Figure 2. Data (triplicate, mean ± SD) are representative of 3 experiments from independent donors with similar results. *P < .05. (C) Released cell-free structures retained the lysosomal dye AO. AO-loaded eosinophils were stimulated with A23187 for 1 hour. The structures (⩽4 μm) in culture medium from different preparations were subjected for flow cytometry. The graph shows the structures from AO-loaded cells (black line); fixed (2% PFO) and permeabilized (0.1% saponin) structures from AO-loaded cells (gray line); and structures from cells without AO staining (filled histogram). Data are representative of 3 experiments from independent donors with similar results. (D) A23187-induced subcellular structures were not apoptotic bodies. Subcellular structures (⩽4 μm) from A23187 and anti-Fas Ab–stimulated cells were stained with annexin V, followed by flow cytometric analysis. Fas-stimulated apoptotic cells produce annexin V–positive subcellular structures (ie, apoptotic bodies, filled histogram), but A23187-stimulated ETosis cells did not (open histogram). Apoptosis was induced, as described in supplemental Figure 4. Data are representative of 3 experiments from independent donors with similar results. (E) Isolated granule-rich subcellular structures retained the granule protein MBP; some were variably associated with PM-derived MHC class I proteins, regardless of size. Culture medium of A23187-stimulated cells was collected, and residual cells were removed by centrifugation (200 × g for 10 minutes). DNA was removed by DNase treatment, and buoyant vesicles and membranes were removed by further centrifugation (2500 × g for 10 minutes). Isolated granule-rich structures were stained for MBP and MHC class I (open histograms). Total structures (⩽4 μm) and small structures (⩽1 μm) were gated. Filled histograms show isotype-matched controls. Data are representative of 3 experiments from independent donors with similar results. Scale bars represent 5 μm (A). NS, not significant.