Fig. 4

NET formation was induced by platelet-derived exosomes during septic shock. Exosomes were isolated from the plasma of healthy controls and septic shock patients. a PMNs isolated from the peripheral blood of healthy volunteers were cocultured with PBS or 100 μg/mL exosomes or 50 nM PMA for 3 h, and NET formation was visualized by confocal microscopy using the following fluorescent dyes: Hoechst 33342 (blue) and SYTOX Green (green). b Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. c PMNs were cocultured with various exosome concentrations for different periods, and the supernatant dsDNA concentration was measured using PicoGreen fluorescent dye. d PMNs were cocultured with exosomes (100 μg/mL) for different periods, and the MPO-DNA complexes were measured using ELISA. e–h Mice were injected with PBS or exosomes (300 μg/mouse, i.p.) isolated from the plasma of sham or CLP mice for 24 h. e, f Quantification of dsDNA and circulating NET structures in the plasma of mice using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively. g Quantification of dsDNA in BALF using PicoGreen fluorescent dye. h Representative images of direct immunofluorescence staining of DNA (blue) and MPO (red) in lung sections after exosome injection. i Ex vivo fluorescence signals in the lungs of mice injected i.p. with Dil-labeled exosomes from equal plasma volumes (100 μL). j–l Platelets were stimulated with PBS or 1 μg/mL LPS or 0.1 U/mL thrombin for 3 h. Exosomes were isolated from the supernatant of stimulated platelets and cocultured with PMNs for 5 h. j NET formation was visualized by confocal microscopy. k, l Quantification of dsDNA and circulating NET structures in the supernatant of cultured PMNs using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively. All results are representative of 3 independent experiments. Graphs represent means ± SEM; **P < 0.01 compared within two groups