Grx1 is a master regulator of actin cytoskeleton rearrangement and DNA release. (A) Confocal microscopy. DNA release was analyzed after short-term stimulation of WT, Nox2^−/−, and Grx1^−/− mouse neutrophils with the indicated triggers in a time-dependent manner. n = 5. (B) Confocal microscopy. DNA-releasing neutrophils were quantified in ten high-power fields in a time-dependent manner (numbers indicated C5a stimulation time in minutes). In contrast to WT mouse neutrophils, both Nox2^−/− and Grx1^−/− neutrophils were completely devoid of any extracellular DNA. n = 5. (C) Flow cytometry. Total ROS activity of WT, Nox2^−/−, and Grx1^−/− mouse neutrophils after short-term stimulation with the indicated triggers was assessed in a time-dependent manner (numbers indicated C5a stimulation time in minutes) by using DHR123 fluorescence. n = 5. (D) Confocal microscopy. F-actin distribution and morphological changes were analyzed after short-term stimulation (total 35 min) of WT and Grx1^−/− mouse neutrophils with the indicated triggers. Right: Quantification of F-actin was performed by automated analysis of microscopic images by using Imaris software. n = 3. (E) Flow cytometry. Expression of CD63 was assessed after pretreatment and short-term stimulation (total 35 min) of WT and Grx1^−/− mouse neutrophils with the indicated triggers. Representative histograms are presented. n = 5. Data are means ± SEM. ***, P < 0.001. Bars, 10 µm.