Coculture system

For coculture experiments, target cells and neutrophils were cocultured at a ratio of 1:1 in the presence of PMA for 15 hours. Subsequently, the cells were harvested by centrifugation at 500g for 10 min at 4°C and lysed in TRIzol. The inhibitors RU.521 (10 μM, InvivoGen), CCCP (10 μM, MedChemExpress), H151 (4 μg/ml, InvivoGen), amlexanox (10 μg/ml, InvivoGen), Pyro (30 μM, Sigma-Aldrich), DPI (100 nM, Sigma-Aldrich), nystatin (10 μg/ml, Sigma-Aldrich), and CytoB (5 μg/ml, Sigma-Aldrich) were added before PMA stimulation was performed. In the Transwell system, cells were separated by a 0.4-μm pore membrane. If NEi (20 μM) was used to inhibit NET formation, then it was directly added at the beginning of the coculture period. If NEi was used to test the requirement of NE for NET phagocytosis, then it was added 2 hours after the cells were stimulated, to enable NET formation. For microscopy, macrophages were seeded in eight-well, glass-bottom dishes (ibidi) and cocultured with neutrophils at a ratio of 1:1 and treated with 100 nM PMA. For live cell imaging, neutrophils were prestained with CellTracker Deep Red Dye (Thermo Fisher Scientific) according to the manufacturer’s instructions. LysoTracker Red DND-99 (50 nM, Thermo Fisher Scientific) and Dextran Alexa 568 (1 μg/ml, Thermo Fisher Scientific) were added before stimulation.