Quantifying Neutrophil Extracellular Traps

NET formation was tested using confocal microscopy as previously described (79 (link)). Sorted CD16^Int (0.5 × 10⁶ cells/well) were resuspended in NET media (colorless RPMI + 0.5% BSA +10 mM HEPES) and seeded onto sterile acid–washed coverslip coated with (1 mg/mL) poly-l-lysine. Cells were incubated for 60 minutes in CO₂ incubator. After incubation, cells were fixed with 2% PFA for 30 minutes, washed twice with PBS, and blocked with 1% BSA in PBS for 1 hour. NETs were determined by extracellular colocalization of anti-human lactoferrin antibody (1:500 dilution, MP Biomedicals, catalog 55040) with DAPI (600 nM for 10 minutes) nuclear stain. The secondary antibody utilized was Alexa Fluor 647 (1:1000; Invitrogen, Thermo Fisher Scientific, catalog A21246). Confocal images and Z-stacks (1 μm thickness for each slice) were obtained by the Fluoview FV1000 confocal microscope (Olympus) with the 63× oil objective. Confocal Z-stack images were used to quantify colocalization of extracellular DNA and lactoferrin using IMARIS v9.6 software (Oxford Instruments).