Inflammasome activation kinetics and localization during NETosis in mouse neutrophils. ASC citrine (green) reporter neutrophils were stained with far-red SiR-DNA (blue) or with far-red SiR-tubulin (red), and time-lapse DIC and spinning-disk confocal images were taken at 2- to 5-minute intervals for 4 hours. (A) Time series of image overlays of DIC (grayscale) and fluorescence of cell and DNA dynamics in neutrophils during NETosis in the presence of LPS. (A and F) Boxes around images indicate different cellular events (orange, speck formation; purple, microvesicle shedding [MV]; green, nuclear rounding; yellow, DNA release to the cytosol; black, extracellular DNA release). (B) Timing of cellular events relative to MV shedding under LPS activation. Each dot represents an individual cell. (C) Arithmetic means of percentage of cells forming ASC speck as a function of time in the absence (black curve) or in presence (red curve) of LPS added at time = 0 minutes. (D) Fluorescence intensity of ASC-citrine speck in neutrophils according to the time relative to MV shedding. Values are means plus or minus SEM. (E) Percentage of NETosis in cells forming speck (•) or in cells not forming speck (▪) under LPS stimulation. Each dot represents 1 independent experiment. Bar size represents the mean of 12 independent experiments. A Mann-Whitney U test was used; ***P < .001. (F) Time series of image overlays of DIC (grayscale) and fluorescence of ASC (green) and MT (red) in mouse neutrophils. White arrows indicate the presence of the ASC speck. Scale bar, 10 μm. MT, microtubules; SEM, standard error of the mean.