Neutrophil extracellular traps (NETs) are extracellular structures that bind and kill microorganisms. Previously, it was believed that NETs are formed by dying or dead neutrophils. However, this study shows that viable neutrophils can generate NETs after priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with toll-like receptor 4 (TLR4) or complement factor 5a (C5a). These NETs contain mitochondrial, but not nuclear, DNA. The formation of NETs is dependent on reactive oxygen species (ROS), and viable neutrophils stimulated with GM-CSF and C5a show increased survival compared to resting neutrophils, which do not generate NETs. This suggests that mitochondrial DNA release and NET formation do not require neutrophil death and do not limit the lifespan of these cells. The study also shows that NETs formed by viable neutrophils do not require cell death, as evidenced by the absence of cell membrane damage and the lack of apoptosis markers. Furthermore, the release of mitochondrial DNA is independent of cell death, as shown by the inability of neutrophils from patients with chronic granulomatous disease (CGD), who have impaired ROS production, to release DNA. The study also demonstrates that the release of mitochondrial DNA is dependent on ROS generation. These findings suggest that NET formation is a cell death-independent process and that viable neutrophils can contribute to innate immunity by releasing mitochondrial DNA.Neutrophil extracellular traps (NETs) are extracellular structures that bind and kill microorganisms. Previously, it was believed that NETs are formed by dying or dead neutrophils. However, this study shows that viable neutrophils can generate NETs after priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with toll-like receptor 4 (TLR4) or complement factor 5a (C5a). These NETs contain mitochondrial, but not nuclear, DNA. The formation of NETs is dependent on reactive oxygen species (ROS), and viable neutrophils stimulated with GM-CSF and C5a show increased survival compared to resting neutrophils, which do not generate NETs. This suggests that mitochondrial DNA release and NET formation do not require neutrophil death and do not limit the lifespan of these cells. The study also shows that NETs formed by viable neutrophils do not require cell death, as evidenced by the absence of cell membrane damage and the lack of apoptosis markers. Furthermore, the release of mitochondrial DNA is independent of cell death, as shown by the inability of neutrophils from patients with chronic granulomatous disease (CGD), who have impaired ROS production, to release DNA. The study also demonstrates that the release of mitochondrial DNA is dependent on ROS generation. These findings suggest that NET formation is a cell death-independent process and that viable neutrophils can contribute to innate immunity by releasing mitochondrial DNA.