Extracellular bacterial killing associated with NET formation is deficient in neonatal PMNs. NET trapping of S aureus (A) and E coli (B) was examined by scanning electron microscopy following incubation of the bacteria with unstimulated PMNs from adult donors for 60 minutes. These images are representative of 3 different experiments. (C) Total bacterial killing by PMNs isolated from adults and term infants following stimulation with LPS (100 ng/mL) for 1 hour was determined (left bars). To determine NET-associated extracellular bacterial killing, phagocytosis and intracellular bacterial killing were blocked by pretreatment with cytochalasins B and D (right bars). DNase treatment was used to degrade NETs formed following LPS stimulation and block NET-associated bacterial killing to thus determine phagocytotic, intracellular bacterial killing (middle bars). See “Bacterial killing assay” for additional details. The bars indicate mean bacterial killing plus or minus SEM in 3 separate experiments. An asterisk indicates a significant difference (P < .05) in total bacterial killing and phagocytotic, intracellular bacterial killing by PMNs isolated from term infants compared with PMNs isolated from healthy adults. A double asterisk indicates a significant difference (P < .001) in extracellular, NET-associated bacterial killing by PMNs isolated from term infants compared with PMNs isolated from adults.