0.4 μm pore size membrane

The concentration of particles was adjusted with DMEM prior to incubation with neutrophils. Neutrophils, either treated with particles for 2 hours at a constant particle-to-cell ratio or not, were co-incubated with THP-1, a human acute monocytic leukemia cell line (American Type Culture Collection; ATCC), either directly or in a Transwell insert for additional 8 hours. Neutrophils were separated from the macrophages in the lower well by a 0.4-μm pore size membrane (Corning). Prior to co-culture with neutrophils, THP-1 cells were cultured at 10⁶ cells/ml in RPMI 1640 medium containing 10% FBS. THP-1 cells were primed with 200 nM PMA (ENZO Life Science) for 24 hours. In some experiments, DNase I (Roche) was used to treat NETs. DNase I was added to culture at a final concentration of 10 units/ml. Recombinant human HMGB1 (PlexBio) was used to stimulate THP-1 for 8 hours at 100 ng/ml as a positive control. Chicken anti-HMGB1 polyclonal antibody was purchased from IBL international. Mouse anti-TLR2 and 4 and anti-RAGE-neutralizing antibodies were obtained from eBioscience and R&D Systems respectively, and used in some co-culture experiments at 10 μg/ml.

HUVECs were seeded on the top Transwell chamber with 0.4 μm pore-size membrane (Corning, 3413) and grown for a minimum 2 days until full confluence. Cells were treated with H₂O₂ (200 μM) with or without PDA (100 mg/mL) for 6 h at 37 °C, followed by 3 washs with PBS. FITC-dextran of 70 kDa (Sigma, 1 mg/mL) was added to the top chamber. After 1.5 h, the sample was collected from the bottom chamber and read in a fluorescence microplate reader (Synergy2, BioTek, Winooski, VT, USA) at 485/528 nm.