Neutrophil defense mechanisms are altered upon actin polymerization inhibition. (A) Extracellular ROS production in response to PMA (100 ng/mL) and serum-opsonized yeast particles (1 mg/mL) were measured by using the Amplex Red assay upon preincubation with the indicated inhibitors (mean ± SD, n = 4-13). (B) Extracellular ROS production in response to opsonized C. albicans and A. fumigatus measured by Amplex Red assay upon preincubation with the indicated inhibitors (mean ± SD, n = 3). (C) 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 of the vehicle control (mean ± SD, n = 4-15). (C) Neutrophil viability upon 4 hours incubation with actin polymerization inhibitors was assessed by flow cytometry (mean ± SD, n = 2-17). (D) Phagocytosis of opsonized A. fumigatus-RFP (yellow) was assessed by live cell imaging. DRAQ5 (cyan) was used as DNA dye. Representative stills after 31 minutes are displayed (see also supplemental Video 2). (E) Chemotaxis upon stimulation with C5a (10 nM) measured over 3 µm pore-size filters (mean ± SD, n = 1-5). Bars represent 50 μm (D). PMNs, polymorphonuclear leukocytes; RFP, red fluorescent protein.