ROSs induce NET formation by adult PMNs but do not complement defective NET formation by PMNs isolated from newborn infants. NET formation was detected by live cell imaging with confocal microscopy (20× objective) using cell-permeant (green fluorescence) and impermeant (red fluorescence) DNA dyes as in Figure 1. All images are representative of 4 different experiments using PMNs from each subject group. (A) PMNs isolated from adults were incubated with buffer alone (control) or stimulated with LPS (100 ng/mL) for 60 minutes. (B) Adult PMNs were pretreated with DPI (20 μM) and then incubated for 60 minutes with either LPS (100 ng/mL) or exogenous ROSs generated by treatment with glucose/GO (1 mM/200 mU/mL). DPI inhibited endogenous ROS generation. (C) Adult PMNs were treated with glucose/GO (1 mM/200 mU/mL) alone without LPS or other agonist stimulation. (D) PMNs isolated from term (left panel) or preterm (right panel) infants were stimulated with exogenous ROSs by treatment with glucose/GO (1 mM/1000 mU/mL). Additional incubations using GO in concentrations from 200 mU/mL (ie, as with adult PMNs in panels B and C) to 1000 mU/mL yielded similar results (not shown).