Actin polymerization requires ROS production and is essential for DNA release. (A) Confocal microscopy. DNA release was analyzed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated triggers in the presence or absence of 1 µM DPI and 1.25 µM Lat B. Right: Quantification of released dsDNA in supernatants of activated neutrophils. n = 5. (B) Confocal microscopy. F-actin distribution and morphological changes were analyzed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors and triggers. Right: Quantification of F-actin was performed by automated analysis of microscopic images by using Imaris software. n = 4. (C) Flow cytometry. Total ROS activity of human neutrophils after pretreatment and short-term stimulation (total 35 min) with the indicated inhibitors and triggers was assessed by DHR123 fluorescence. n = 5. (D) Flow cytometry. Expression of CD63, CD66b and CD35 was assessed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors, DPI (5 µM), and Lat B (10 µM), and triggers. Representative histograms are presented. n = 5. Data are means ± SEM. ***, P < 0.001. Bars, 10 µm.