Quantitation of NET formation by MPO-deficient neutrophils. Nuclear areas of neutrophils stimulated with PMA as in Figure 1 are plotted against the percentage of Sytox-positive cells corresponding to a given nuclear area range. Cells were unfixed (A-B,E) or fixed with paraformaldehyde (C-D) before Sytox staining. (Ai) By 4 hours after PMA stimulation, neutrophils from a control donor made NETs, indicated by the broad range of nuclear areas. Naive cells were still alive and did not stain with Sytox. (Aii) In contrast, neutrophils from a completely MPO-deficient donor did not form NETs. Instead, the cells died by 6 to 8 hours after PMA stimulation, but their nuclei remained small. (B-C) Neutrophils from 2 other completely MPO-deficient donors did not make NETs after 4-6 hours of PMA stimulation. (D) Neutrophils from 3 partially MPO-deficient donors made NETs. (E) Neutrophils from a control donor pretreated with the MPO inhibitor ABAH displayed a 2-hour delay and significant decrease in NET formation, reaching peak nuclear areas at 6 hours instead of 4 hours after PMA stimulation. Color scheme: gray, naive cells; black, control or untreated cells; red, completely MPO-deficient cells; blue, partially MPO-deficient cells; green, ABAH-treated cells. Statistical analysis: ***P < .0001; n.s., differences not significant. (A) Control versus donor 1 (6 hours)***; control versus donor 1 (8 hours)***; donor 1 (6 hours) versus donor 1 (8 hours), n.s. (B) Control versus donor 2***. (C) Control versus naive***; control versus donor 3***; donor 3 versus naïve, n.s. (D) Control versus donor 4***; control versus donor 5, n.s.; control versus donor 6***. (E) 4-hour PMA versus 4-hour PMA plus ABAH***; 4-hour PMA versus 6-hour PMA plus ABAH***; 4-hour PMA plus ABAH versus 6-hour PMA plus ABAH, n.s. For each sample, 100-500 cells were evaluated.