Immunofluorescence Visualization of NETs

The protocol for NET visualization by immunofluorescence was as previously described (15 (link), 21 (link)). Briefly, 2 × 10⁵ cells in RPMI-1640-0.5% FBS were seeded onto a round glass coverslip in a 24-well plate. Cells were allowed to adhere for 10 min before addition of PMA at various concentrations for 2 h (37°C, 5% CO₂). Cells were then fixed with 4% paraformaldehyde, and were permeabilized with PBS containing Triton-X 100 (0.5%). Nuclear morphology and NETs were stained using Sytox Green (1 μM, ThermoFisher, MA, USA), Hoechst 3342, or Hoechst and an anti-MPO antibody (Santa Cruz, sc-52707, 1:500). Cells were mounted onto slides, and were observed using a Nikon Eclipse 80i microscope. The percentage of NET formation was quantified by counting cells with decondensed/rounded nuclei and extracellular Sytox Green positive fibers in at least three randomly pictured fields.