Inhibition of myosin II results in impaired NETosis. (A) Extracellular ROS production in response to PMA (100 ng/mL) and serum-opsonized yeast particles (1 mg/mL) were measured by Amplex Red assay upon preincubation with pa-bleb (mean ± SD, n = 3. (B) Protease release upon stimulation with C5a (10 nM), N-formyl-Met-Leu-Phe (fMLF) (1 µM), and CytoB/fMLF (5 µg/mL/1 µM) was determined by DQ-green BSA. A Triton X-100 lysate was used as 100%. Results are shown relative to the Triton-X lysate (mean ± SD, n = 3-4). (C) Chemotaxis upon stimulation with C5a (10 nM) measured over 3-µm pore-size filters (mean ± SD, n = 3). (D) Phagocytosis of opsonized C. albicans was assessed by live cell imaging. DRAQ5 (cyan) was used as DNA dye. Representative stills after 33 minutes are displayed. White arrows indicate C. albicans conidia inside the neutrophil; black arrow indicates a long uropod indicative of impaired uropod retraction. (E) Neutrophils were preincubated with pa-bleb or with the vehicle control and NET formation was induced by PMA for 4 hours at 37°C. NETs were visualized by staining for NE (green), MPO (magenta), and DNA (Hoechst, cyan). Images were acquired by using a Leica SP8 confocal microscope. (F-G) DNA release in response to PMA (mean ± SD, n = 4-7) (F) or opsonized C albicans (mean + SD, n = 2) (G) was measured in real time by Sytox Green–fluorescence, and the area under the curve (AUC) was calculated relative to the vehicle control (mean ± SD). Bars represent 25 μm (D) and 100 μm (E). **P < .01; ****P < .0001. RFU, relative fluorescence unit.