Quibit 2.0 fluorimeter

Extracellular DNA traps were quantified in BALF cells supernatants stimulated with RSV (10³ PFU/mL) or unstimulated using Quant-iT ds DNA HS (Invitrogen, Carlsbad, CA, USA) and measured in the Quibit 2.0 fluorimeter (Invitrogen), according to the manufacturer's recommendations. Visualization of EETs by fluorescence microscopy BALF cells (2 × 10⁵/mL) were stimulated with phorbol-12-myristate-13-acetate (50 nM). After that, cells stimulated with RSV (10³ PFU/mL) and unstimulated ones were incubated for 3 hours, fixed with 4% paraformaldehyde and stained with anti-eosinophil peroxidase (EPO) and anti-histone H2B (1:250; Santa Cruz Biotechnology, Dallas, TX, USA) for 45 minutes. Later, cells were incubated with fluorescein isothiocyanate (1:100; Santa Cruz Biotechnology) and alexa fluor 633 (1:100; Invitrogen) for 30 minutes. Next, the cells were stained with Hoechst 33342 DNA dye (1:2,000; Invitrogen) for 4 minutes. Confocal images were taken on a Leica TCS-SP8 exciter microscope (Leica Microsystem, Wetzlar, Germany).