Fig. 3.

Ex vivo NETosis assay. Mice were administered LPS and euthanized 12 h later for bronchoalveolar lavage (BAL). A: fluorescent images of BAL neutrophils undergoing NETosis. Unstimulated neutrophils stained with Sytox Orange demonstrated characteristic multilobed nuclei. Neutrophils incubated with 1 μM phorbol myristate acetate (PMA) or 100 multiplicity of infection (MOI) live Pseudomonas aeruginosa (PsA) for 2.5 h formed abundant neutrophil extracellular traps (NETs), as indicated by extracellular DNA staining. B: kinetic fluorescence assay for quantifying NETosis from BAL neutrophils. Lysis with saponin defined maximal DNA release, whereas stimulation with PMA or P. aeruginosa stimulated NETosis. P. aeruginosa itself spontaneously released low levels of DNA. C: DNA release in response to PMA was dose-dependently inhibited by the NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI). Error bars represent SE.