Patients with ARPC1B and MKL1 deficiency are impaired in NET formation. Representative images of PMA-induced (A), C. albicans–induced (B), and MSU crystal–induced (C) NETs by control and ARPC1B-deficient neutrophils visualized by staining for NE (green), MPO (magenta), and DNA (cyan; Hoechst). Arrows indicate NET structures around C. albicans hyphae and MSU crystals (control) or neutrophils adhered/engulfing C. albicans hyphae or MSU crystals (patient). Images were acquired by using a Leica SP8 confocal microscope. (D) DNA release induced by MSU crystals was visualized by Sytox Green. Images were acquired by an EVOS Fluorescence Cell imaging system. (E) DNA release was measured in real-time by Sytox Green–fluorescence for 4 hours at 37°C, and the area under the curve (AUC) was calculated relative to the relevant day control neutrophils (mean ± SD, n = 6-14). (F) Quantification of NETosis by control and ARPC1B-deficient neutrophils (n = 3) determined as relative number of decondensed nuclei after 4 hours activation with PMA. (G) PMA-induced NETs by control and MKL1-deficient neutrophils (n = 1). Bars represent 100 μm (A-C), 400 µm (D), and 100 µm (G). DIC, differential interference contrast.