Neonatal NET formation. (A) Kinetics of NET formation by neonatal and adult neutrophils. Neutrophils were isolated from cord blood (n = 3) or peripheral adult blood (n = 3) by Percoll gradient centrifugation and erythrocyte lysis. Isolated neutrophils were stimulated with Pam3CSK4 (1 μg/mL), heat-killed Listeria monocytogenes (HKLM, 108/mL), FSL-1 (1 μg/mL), LPS (1 μg/mL), flagellin (1 μg/mL), ssRNA40 (1 μg/mL) or ODN2006 (2.5μM) for 1, 2, or 3 hours at 37°C. Afterward, NETosis was analyzed by DAPI (DNA staining) and citrullinated histone 3 staining and confocal microscopy. The percentage of NET-producing neutrophils was calculated from all neutrophils analyzed. *P < .05, neonatal versus adult NETosis. (B) Confocal laser scanning microscopy images of TLR-induced NETosis. Neutrophils were stimulated for 2 or 3 hours at 37°C with LPS (1 μg/mL). Blue channel indicates DNA (stained with DAPI) in stimulated neutrophils isolated from newborn and adult blood. (C) Scanning electron microscopy of neonatal NETosis. NETosis in neonatal neutrophils after LPS stimulation (1 μg/mL, 3 hours, 37°C) at 3 different magnifications. (D) Confocal laser scanning microscopy of histone citrullination. Neonatal neutrophils were stimulated with 1 μg/mL LPS for 3 hours at 37°C. Blue channel indicates DAPI; red channel, citrullinated histone 3; and green channel, F-actin. (E) Scanning electron microscopy of bacteria-induced NETosis. NET formation by neonatal neutrophils after coincubation with nonmucoid Pseudomonas aeruginosa (PAO.1) for 3 hours at 37°C. (F) Confocal light scanning microscopy of bacteria-induced NETosis. Neonatal neutrophils coincubated with nonmucoid P aeruginosa (PAO.1) for 3 hours at 37°C. Live/dead staining (BacLight bacterial viability kit, Invitrogen). Red channel indicates propidium iodide staining of extracellular DNA (NETs) and dead bacteria; green channel, Syto-9 stains live bacteria and intracellular DNA in the nuclei of living neutrophils; and yellow-red, overlay (dead bacteria). (B,D,F) Images were captured using an Olympus IX81 microscope (Fluoview 1000) and an Olympus XC30 camera, with Olympus Fluoview Software FV10-ASW 0.200, a 60×/1.35 oil objective, and Vectashield Mounting Medium. (C,E) Images were captured using an ESEM XL30 microscope (FEI, Philips; 20 kV).
