NET formation and inhibition of A nidulans growth. Control (A,D), but not CGD (B,E), neutrophils made NETs on 3-hour PMA stimulation. For immunofluorescence, NETs were stained with an antibody that recognizes neutrophil elastase (green; A-C). NETs were clearly visible also by scanning electron microscopy (SEM; D-F). Neutrophils isolated from the CGD patient before GT could be activated because they flattened out (E) but did not make NETs. The ability to form NETs was partially restored by GT 6 weeks after GT (C,F white arrows). (G) Quantification of NET-DNA released after 3 hours of PMA stimulation of control neutrophils, CGD neutrophils before and 6 weeks after GT or (H) after stimulation of CGD gp91^phox+ and CGD gp91^phox− FACS-sorted neutrophils. CGD gp91^phox+ neutrophils showed normal NET formation, whereas CGD gp91^phox− neutrophils showed only residual NET formation. FACS-sorting efficiency was 90% to 92% for CGD gp91^phox− and 95% to 96% for CGD gp91^phox+ cells. (I-J) NET inhibition of A nidulans conidia and hyphae. (I) Conidia were plated on FACS-sorted neutrophils prestimulated with PMA plus or minus MNAse (ie, when NET formation was complete, cells were dead and therefore incapable of phagocytosis). Hyphal outgrowth was measured after 16 hours. (J) Hyphae were coincubated with FACS-sorted neutrophils, and PMA plus or minus MNAse and hyphal viability was assessed after 5 hours. (G-J) Data are mean ± SD of a representative triplicate experiment. Inhibition of fungal growth is expressed as percentage of control values (A nidulans conidia or hyphae incubated in media). The differences between −MNase and +MNase were significant (for control and CGD gp91^phox+ cells) by Student t test: **P < .01; ***P < .001.