Actin cytoskeleton rearrangements are required for NET formation in response to PMA. Neutrophils were preincubated with the indicated actin polymerization inhibitors, or with the vehicle control, and NET formation was induced by PMA for 4 hours at 37°C. (A) NETs were visualized by staining for NE (green), MPO (magenta), and DNA (cyan; Hoechst). Images were acquired by using a Leica SP8 confocal microscope. (B) DNA release was measured in real-time by Sytox Green–fluorescence. (C) The area under the curve (AUC) was calculated relative to the vehicle control (mean ± SD, n = 6-19). (D) Zoom of panel A. Neutrophil nuclei morphology after 4 hours of PMA stimulation was examined. DNA was visualized (cyan; Hoechst). Images were acquired by using a Leica SP8 confocal microscope. The mixed effects model with Dunnett’s test for multiple comparisons was used to test statistical significance. ****P < .0001. Bars represent 50 μm (A) and 25 µm (D). ns, nonsignificant; RFU, relative fluorescence unit.