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. 2016 Nov 4;7:484. doi: 10.3389/fimmu.2016.00484

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Copyright © 2016 Pieterse, Rother, Yanginlar, Hilbrands and van der Vlag.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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All LPS structures induce “vital” NETosis in the presence of platelets. (A) Typical extracellular DNA filaments (white arrows) were observed in whole blood cultures ex vivo after 180 min of incubation with the different LPS serotypes. (B) Platelets (panel 2) or LPS-O111 (panel 3) alone does not induce NETosis after 180 min of incubation with neutrophils, whereas the combination of both (panel 4) stimulates NET formation without neutrophil (lytic) cell death. Massive neutrophil cell death is observed in response to LPS-PA alone (panel 5), based on the failure to exclude the vital dye Sytox Orange (yellow), which can be largely prevented by the addition of platelets (panel 6). LPS was used at a concentration of 8 pg LPS per neutrophil. Notably, representative light microscopy images are shown to visualize neutrophil morphology after stimulation and do not correspond in terms of “field of view” to the adjacent representative immunofluorescence images. Scale bars: white = 20 μm and red = 40 μm.