Exogenous addition of H₂O₂ restored the ability of Nox2^−/− mouse neutrophils to perform actin polymerization, to degranulate, and to release DNA. (A) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of WT and Nox2^−/− mouse neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Right: Quantification of released dsDNA in supernatants of activated neutrophils. RFU, relative fluorescence units. (B) Confocal microscopy. F-actin distribution and morphological changes were analyzed after short-term stimulation (total 35 min) of WT and Nox2^−/− mouse neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Right: Quantification of F-actin was performed by automated analysis of microscopic images by using Imaris software. AU, arbitrary units. (C) Flow cytometry. Expression of CD63 was assessed after pretreatment and short-term stimulation (total 35 min) of WT and Nox2^−/− mouse neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Representative histograms are presented. (D) Bacterial killing by colony formation unit (cfu) assay. E. coli killing was analyzed after short-term stimulation (total 35 min) of WT and Nox2^−/− mouse neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Data are means ± SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05. n = 5.