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Significance Neutrophils are white blood cells specialized as the first line of host defense in the immune system. One way they protect organisms is through NETosis, in which they expel their DNA to form a web-like trap that ensnares pathogens and promotes clotting. However, NETs also mediate sterile inflammation, causing damage to the body. We used high-resolution live-cell microscopy to characterize the timing of dynamic cellular events leading to NETosis in human and mouse neutrophils and a neutrophil-like cell line. We discovered that NETosis proceeds by a stepwise sequence of cellular events that is conserved across species and requires the activity of the PAD4 enzyme for DNA to be released from the nucleus and cell membrane.

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture