Stimulated PMNs isolated from newborn term or preterm human infants fail to form NETs. (A) NET formation was detected by live cell imaging with confocal microscopy using 2 DNA dyes, one cell impermeable (Sytox Orange, which stains extracellular DNA red in these images) and the other cell permeable (Syto Green, which identifies nuclear DNA) after activation of PMNs with LPS (100 ng/mL) for 60 minutes. Experiments were performed on poly-l-lysine– or fibrinogen-coated (top row, third panel) glass coverslips. DNase (2.5 Units/mL) was added following LPS stimulation (60 minutes) to dismantle extracellular NETs (top row, far right panel). The images are representative of experiments performed with LPS-stimulated PMNs isolated from 6 different donors from each subject group (adult, term, and preterm). Incubations on immobilized fibrinogen and treatment of LPS-stimulated PMNs with DNase were performed with neutrophils from 3 different adult subjects in each case. Control and LPS stimulation, 20× objective. (B) NET formation was detected as in panel A following stimulation of adult, term, and preterm PMNs with PAF (10 nM) for 60 minutes. These images are representative of separate experiments using PMNs isolated from 6 different donors in each subject group (adult, term, and preterm). Control and PAF stimulation, 60× objective. (C) NET formation was examined by scanning electron microscopy following stimulation of PMNs with LPS (100 ng/mL) for 60 minutes. These images are representative of 3 different experiments with PMNs from different donors in each subject group.