Fig. 3

ROS support enhanced NETosis in AOSD patients [39]. Cytoplasmic total reactive oxygen species (ROS) stained with DCFH-DA after the activation of neutrophils with 20 nM phorbol myristate acetate (PMA) for 3.5 h was detected using flow plates (a) and flow cytometry (b and c); the results show the relative fluorescence intensity, ROS-positive neutrophils, and a representative histogram. Mitochondrial ROS production in neutrophils stimulated with 20 nM PMA for 3.5 h and stained with the red fluorescent mitochondrial superoxide indicator, Mito SOX, was determined using flow cytometry (d) and immunofluorescence (e). Neutrophils from subjects with adult-onset Still’s disease (AOSD; n = 10) were stimulated with 20 nM PMA in the presence of the indicated ROS inhibitor. f,g Sytox green immunofluorescence analysis was performed, and images of neutrophil extracellular trap (NET) formation from each culture were obtained. h The quantification of the DNA concentration was achieved using PicoGreen. The figures are representative of six independent experiments. Scale bars = 20 μm. The histograms show the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. HC healthy controls