Slide flask chambers

Purified neutrophils (10 × 10⁵ cells per cm²) were seeded in slide-flask chambers (Thermo Scientific) and exposed to MPs. After stimulation, cells and NETs were fixed in 4% paraformaldehyde (15 min, 4°C), and slides were stained for DNA (Sytox Orange; 200 nM), NE (1:200, Abcam), and/or MPO (1:400, Biolegend). Goat anti-rabbit and goat anti-mouse antibodies labeled with Alexa Fluor 488 (1:200, Thermo Fisher Scientific) were used to detect anti-NE and anti-MPO, respectively. Slides were embedded in Vectashield Mounting Medium (Brunschwig Chemie), and pictures were obtained with a Zeiss fluorescence microscope with Axiovision soſtware.

Purified neutrophils (3 × 10⁵ cells per cm²) were seeded in slideflask chambers (Thermo Scientific, Cat. No. 170920, Duisburg, Germany) and stimulated with LPS from different bacterial sources, at the indicated concentrations and conditions, for 3–5 h at 37°C. Where indicated, stimulation of neutrophils with 100 nM PMA served as a positive control. After stimulation, cells and NETs were fixed in 4% paraformaldehyde (30 min, room temperature), and slides were stained for DNA (Sytox Orange; 100 nM), NE (dilution 1:200; antibody listed above), and/or MPO (dilution 1:100; antibody listed above). Slides were embedded in Vectashield Mounting Medium (Brunschwig Chemie, Cat. No. H-1200, Amsterdam, The Netherlands), and pictures were obtained with a Zeiss fluorescence microscope with Axiovision software (Sliedrecht, The Netherlands).