Revolution spinning disk confocal system

Prior to incubation or stimulation, isolated neutrophils were seeded in lysine-coated glass coverslips (Neuvitro; H-12-1.5-pdl). Fixation was performed using 4% paraformaldehyde (Scharlab; FO00102500) for 60 min at room temperature. Non-specific binding sites were blocked with 5% goat serum (Invitrogen; PCN5000) in PBS 1x. Samples were stained using mouse monoclonal anti-LL-37 antibody (Santa Cruz; sc-166770; 1:200) or mouse monoclonal anti-IL-17 antibody (R&D SYSTEMS; MAB3171; 1:200) and rabbit polyclonal anti-NE antibody (Santa Cruz; sc-25621; 1:200). Following three washes with PBS 1x, a polyclonal goat anti-rabbit Alexa Fluor 647 antibody (Invitrogen; A-21244; 1:500) was utilized as secondary antibody for rabbit polyclonal anti-NE. Following three washes with PBS 1x, a polyclonal rabbit anti-mouse Alexa Fluor 488 antibody (Invitrogen; A-11059; 1:500) was utilized as secondary antibody for mouse monoclonal anti-LL-37 or mouse monoclonal anti-IL-17. DAPI (Sigma-Aldrich; D9542; 1:) was used for DNA counterstaining at a final concentration of 1 μg/ml. Visualization was performed in a confocal microscope (Revolution spinning disk confocal system; Andor, Ireland) with PLAPON 60xO/TIRFM-SP [numerical aperture [NA], 1.45] objective (Olympus).

For the TIRF experiments, a Nikon revolution spinning disk confocal system equipped with a TIRF module and Andor iXON EMCCD camera was used. The images were acquired using 405‐, 488‐, 561‐, and 642‐nm lasers, a 100× objective (NA 1.49), and a 1.5× projection lens, and analyzed using Fiji software. The depth of TIRF field was maintained to 120–200 nm, as measured by optically resolved fluorescent beads.