Saliva induces NETs in the oral cavity. Immunofluorescence microscopy was performed with staining for DNA (DAPI, blue) and for elastase and MPO (both red). All images were acquired using a Nikon Eclipse TE200 equipped with a Hamamatsu C4742-95 CCD camera, using Plan Apochromat 20× and 100× objectives and NIS-elements 3.1 (Nikon) software was used for image acquisition and processing. All images for all figures were acquired and formatted in the same fashion unless stated otherwise. (A) Immunofluorescence microscopy of neutrophils collected from morning saliva demonstrated the presence of NETs judged from presence of extracellular DNA with bound elastase and MPO. Stimulation of neutrophils with saliva caused similar NET formation. The presence of NETS in morning saliva were investigated in 6 donors (5 nonsmokers and 1 smoker) with no systemic disease or known oral or dental disease. NETs were found in all donors with no apparent difference between the smoker and the nonsmokers. Scale bars represent 10 μm. (B) Neutrophils from a 13-month-old patient with LAD1 and a healthy control were stimulated for 1 hour with either saliva or S. aureus or left nonstimulated in RPMI 1640 with HSA. Scale bars denote 200 μm and the error bars denote the difference between 20 randomly selected images. Due to the lack of integrins the neutrophils in this experiment are not stimulated on coverslips but in solution. This gives a lower amount of NETosis in the nonstimulated neutrophils compared with the other experiments in which the neutrophils adhere to coverslips before stimulation. (C) Neutrophils were stimulated with saliva or protein-free saliva. Results from 3 independent experiments are shown (in A and C). Detailed statistical analyses for all figures are included in supplementary Appendix A. Scale bars, 100 μm. **P < .01, columns denote average values and error bars denote standard deviations.