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. 2007 Jan 15;176(2):231–241. doi: 10.1083/jcb.200606027

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Copyright © 2007, The Rockefeller University Press

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Figure 2. — Disintegration of the nucleus and granules allows the formation of NETs. Transmission electron microscopy (a–h) and confocal immunomicroscopy (i–l) of neutrophils. Cells were incubated for 180 min in medium without stimulation (a, e, and i) or activated with 20 nM PMA for 60 (b, f, and j), 120 (c, g, and k), or 180 min (d, h, and l; all incubated for a total of 180 min). e–h are higher magnifications of a–d. (a and e) Naive neutrophils show a lobulated nucleus with clearly defined eu- and heterochromatin and numerous cytoplasmic vacuoles, even after 180 min of incubation. (i) Immunofluorescence staining for nuclear (red, histone–DNA complex antibodies) and granular components (green, neutrophil elastase antibodies) is clearly separated in naive neutrophils. Granules are distributed through the globular cell and exhibit a patchy cytoplasmic pattern in this projection of a confocal z stack. (b) After 60 min of stimulation, nuclei are less lobulated. (f) The space between inner and outer nuclear membrane is dilated. (j) Nuclei and granules are now individually visible because the cell has flattened out. (c) After 120 min, most nuclei no longer show separation of eu- and heterochromatin, and in some cells the nuclear envelope starts to disintegrate into a chain of vesicles surrounding the DNA (arrows). (g) DNA and cytoplasm are no longer separated by a membrane (arrows). (k) Cells lose their distinct granule pattern and show colocalization (yellow) of neutrophil elastase (green) and chromatin (red), especially at the nuclear periphery (arrows). (d) Most neutrophils stimulated for 180 min are nearly entirely filled with decondensed chromatin. (h) Small areas are occupied with residual granules and numerous vesicles. (l) Some cells already released NETs. (m) Extracellular neutrophil elastase was measured as described in Materials and methods. By 180 min, most of the neutrophil elastase was bound to chromatin (gray). The timecourse of release correlates with NET-DNA release (black). The experiment was repeated at least three times with neutrophils from independent donors, with similar results. The data shown is a representative triplicate experiment and presented as a mean value ± the SD. Bars: (a–d) 2 μm; (e–h) 1 μm; (i–l) 10 μm.