Pharmacologic inhibition of actin cytoskeletal rearrangements in human primary neutrophils. (A-B) NETosis dynamics upon PMA stimulation were assessed by live cell imaging. (A) Permeable DNA dye DRAQ5 (cyan) and the impermeable DNA dye Sytox Green (magenta) were used. Arrows indicate the moment neutrophil rounding was observed. Time is displayed in hours. Representative stills are displayed (see also supplemental Video 1). (B) Quantification of the number of neutrophils undergoing NETosis, which showed cell rounding and cell contraction. (C) Live cell imaging of NETosis dynamics upon PMA stimulation using CellMask Orange Plasma membrane Stain (yellow) to visualize the plasma membrane and DRAQ5 (cyan) as DNA dye. Drawing of the cell border is inserted to visualize reduction of cellular surface. Time is displayed in hours. Representative stills are displayed. (D) Schematic overview to illustrate where the pharmacologic actin inhibitors target F-actin assembly. → stabilization or induction, ─| inhibition. (E) Actin polymerization was examined by confocal analysis with staining for F-actin (green = F-actin; phalloidin, cyan = DNA; Hoechst) upon 10 minute stimulation with C5a. Images were acquired by using a Leica TCS SP8 confocal microscope. (F) Actin polymerization upon preincubation with the actin rearrangement inhibitors was examined in suspension after stimulation with C5a (10 nM) by flow cytometry (mean ± SEM, n = 4-7). Bars represent 25 μm (A, C, and E). MFI, mean fluorescence intensity.