Formation of MCETs is dependent of reactive oxygen species (ROS)-induced MCs death. (A) Production of ROS by S pyogenes–infected MCs in the presence or absence of NADPH oxidase inhibitor DPI. Uninfected MCs (i) or S pyogenes–infected MCs cultured in the absence (ii) or presence of DPI (iii) were incubated with NBT for 45 minutes and examined by light microscopy. Precipitation of formazan indicating active production of ROS is indicated by arrows in panel ii (bars, 10 μm). (B) Immunofluorescence photograph showing viable (green) versus dead (red) MCs of uninfected cells (i) or cells after coculture with S pyogenes (ii). Note the release of DNA by dying MCs (arrow) in panel ii. All bars in panel B represent 10 μm. (C) Quantification of MC death in control medium (□), coculture with S pyogenes (■), or coculture with S pyogenes in the presence of DPI (▩). The data are presented as percentage of PI-stained dead cells determined by flow cytometry. Bars represent the means plus or minus SD of 3 independent experiments. *P < .05 by F-test. (D) Quantification of DNA release by MCs after 1 hour of culture in the presence of PMA or S pyogenes and with or without DPI. The amount of DNA was measured as intensity/total flux of red fluorescence (Sytox orange) using the Xenogen Vivo Vision IVIS 200 System with the filter setting of 532 nm excitation/580 nm emission and Igor Pro 4.09A software. A diagram showing the quantitative data are displayed in panel E. (F) FESEM image of MCETs produced by human MCs after 6 hours of either stimulation with 100 mU/mL of glucose oxidase (F) or with S pyogenes (G). Bar, 50 μm.