Poly l lysine coated slides

PBNs isolated from asthmatic patients and HCs were untreated or treated with 100 ng/mL LPS for 2 h for ELISA. In some experiments, PBNs were primed with anti-S100A9 antibodies for 30 min and treated with 100 ng/mL recombinant human S100A9 or LPS for the indicated time (for western blot analysis), 6 h (for confocal microscopy analysis) or 24 h (for cytokine measurement by ELISA).
To detect NET formation by using immunofluorescence, 4 × 10⁶ cells were seeded on poly l‐lysine‐coated slides (Polysciences, Warrington, PA, USA) and then treated with or without the indicated reagents for 6 h. The sections were incubated overnight with anti-myeloperoxidase (MPO), anti-S100A9 (Abcam), and anti-neutrophil elastase (NE) antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit and 594-conjugated donkey anti-goat antibodies (ThermoFisher Scientific) for 1 h. The slides were incubated with DAPI (1:1.000) for 5 min. The images were taken with a confocal laser scanning microscope (LSM710, Cal Zeiss Microscopy GmbH, Jena, Germany)

Peripheral eosinophils were isolated from asthmatic patients as previously described [20 (link)]. To stimulate eosinophils, the cells (1×10⁶) were seeded on a 24-well plate and maintained in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2% fetal bovine serum (FBS; ThermoFisher Scientific). Then, the cells were treated with human recombinant 10 ng/mL IL-5 (Sigma-Aldrich) and 5 ng/mL TGF-β1 (R&D systems). To investigate the effect of cysLTs on eosinophil degranulation, the cells were treated with LTE₄ (Cayman Chemical, Ann Arbor, MI, USA) for 4 hours in the presence of 10 ng/mL IL-5 (Sigma-Aldrich). The function of montelukast (Sigma-Aldrich; 0.1 and 1 μM) against LTE₄ was also investigated. To confirm eosinophil degranulation, eosinophils were seeded on Poly L-lysine-coated slides (Polysciences, Warrington, PA, USA). Then the cells were incubated overnight with anti-eosinophil peroxidase antibody (Cell Signaling, Minneapolis, MN, USA), followed by Alexa fluor 488 donkey anti-rabbit (ThermoFisher Scientific) for 1 hour. 4’,6-diamidino-2-phenylindole (Sigma-Aldrich), and was observed using a Zeiss LSM710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).