pPMNs display surface markers of NETotic DNA. (A) Surface expression of H3Cit, neutrophil elastase (NE), MPO, and the cell-impermeable DNA dye SYTOX were determined on gated rsPMNs (rs) and pPMNs (p) in human blood. FMO controls are indicated. (B) Human blood PMNs were labeled with H3Cit (AF488) and pPMNs were purified by FACS. Cytospins were labeled with DAPI and imaged by fluorescence microscopy. An asterisk (*) indicates surface H3Cit labeling. Representative images are shown. Scale bar, 10 µm. (C) Surface expression of H3Cit, NE, and SYTOX were determined on gated mouse rsPMNs (rs) and pPMNs (p). FMO controls are indicated. (D) H3Cit labeling was performed and cytospins were prepared and labeled with DAPI. PMNs were identified by nuclear morphology. An asterisk (*) indicates surface H3Cit labeling. Representative images are shown. Confocal images were acquired using a Zeiss Axio Observer Z1 microscope with a Plan-Apochromat 63×/1.4 oil-immersion objective. Scale bar, 10 µm. (E) Blood was recovered by cardiac puncture 30 minutes after tail vein injection of SYTOX Green dye (50 µL of 5 µM solution). Representative SYTOX histograms of gated rsPMNs (rs), pPMNs (p), and FMO controls are indicated. (F) SYTOX-labeled NETotic DNA (+sD, 200 ng) or unlabeled DNA (−sD) was added to mouse blood for 10 minutes at 37°C. Gated rsPMNs (rs) and pPMNs (p) were analyzed by flow cytometry. Triplicate results are compiled from 3 independent experiments (n = 3). gMFI of SYTOX signal (mean ± standard error of the mean [SEM]) is shown. P values were determined by ANOVA with a post hoc Fisher's LSD test. ***P < .0005.