Hyperglycemia triggers neutrophil activation and NETosis in cocultures of isolated control neutrophils and BeWo trophoblast cells. (A) Immunofluorescence staining for neutrophil elastase (NE) (red), citH3 (green), and DNA (blue) in polymorphonuclear neutrophil (PMN)–BeWo cell coculture after incubation of PMNs for 3 h with BeWo cells treated with normal glucose (7 mM), high glucose (25 mM), and infliximab (5 µg/ml) for 12 h. Scale bars: 50 µm. (B,C) Morphometric analysis of extracellular citH3+ and NE (NE+/DAPI+) in neutrophils (suggestive of activated and NETing neutrophils) as in (A). (D) Western blot and densitometric analysis of IRS1, GLUT4 and β-actin protein expression levels in BeWo cells incubated with NE (80 nM) and/or insulin (100 nM) (E) Radiometric Glucose Uptake in BeWo cells incubated as in (D). RFU, relative fluorescence units; AU, arbitrary units. Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001) (one-way ANOVA followed by Tukey’s multiple comparison post-test). All experiments were performed at least four times in triplicates with consistent results.