NET formation in response to C. albicans and MSU crystals is reduced when actin dynamics are inhibited. Neutrophils were preincubated with the indicated actin polymerization inhibitors or with the vehicle control, and NET formation was induced by opsonized C. albicans (multiplicity of infection, 10:1) or MSU crystals for 4 hours at 37°C. DNA release was measured in real-time by Sytox Green–fluorescence (A-B) and the area under the curve (AUC) was calculated relative to the vehicle control (C) (mean ± SD, n = 5-7). (D) NETs in response to opsonized C. albicans were visualized by staining for NE (green), MPO (magenta), and DNA (Hoechst, cyan). Arrows indicate NET structures around C. albicans hyphae. Images were acquired by using a Leica SP8 confocal microscope. (E) DNA release induced by MSU crystals was visualized by Sytox Green. Images were acquired by an EVOS Fluorescence Cell imaging system. (F) Quantification of the number of neutrophils bound to C. albicans hyphae upon preincubation with the actin rearrangement inhibitors (mean ± SD, n = 3). The mixed effects model with Dunnett’s test for multiple comparisons was used to test statistical significance. Bars represent 50 μm (D) and 200 μm (E). *P < .05, **P < .01, ****P < .0001. ns, nonsignificant; PMNs, polymorphonuclear leukocytes; RFU, relative fluorescence unit.