DNAse treatment of mDC-PMN coculture abrogates DC uploading with neutrophil proteins. (A) Quantification of NET formation by agar-elicited neutrophils seeded onto poly-D-lysine–coated glasses in the presence of DNAse. Nuclear areas of PMNs are plotted against the percentage of SYTOX-positive cells corresponding to a given nuclear area. In the presence of DNAse, nuclear areas appear smaller and show a narrow peak compared with untreated PMNs in which nuclear area has a broad distribution. Number of nuclei analyzed/experiment = 200. One representative experiment of 3 performed. (B) IF analysis of agar-PMN seeded onto poly-D-lysine-coated glasses in the presence of DNAse (100 U/mL) and DNA dye SYTOX green. Twenty-four hours later, PMNs were fixed and stained with a polyclonal Ab to elastase. Scale bars represent 10 μm. One representative experiment of 3 performed is shown. (C) Forward scatter (FSC)/side scatter (SSC) and annexin V/7-AAD representative plots for CD11b⁺GR-1⁺ agar-PMNs treated or not with DNAse, showing the presence of annexin V⁺ apoptotic cells in agar-PMNs that have been cultured onto poly-D-lysine–coated glasses also in the presence of DNAse. (D-F) Incorporation of PR3 and MPO in mDCs was prevented by DNAse treatment. (D) Confocal microscopy analysis of mDCs (PKH-26⁺, red) cocultured o/n with NETotic PMN in absence (left panels) or presence (right panels) of DNAse and Abs to PR3 or MPO conjugated with Alexa-488 dye. The vital DNA dye Draq 5 (blue) shows that only the DNA of PMNs but not of DCs is degraded by DNAse. Notably, PMNs treated with DNAse retain both PR3 and MPO staining. Scale bars represent 10 μm. One representative experiment of 5 performed. Quantification of PR3 (E) and MPO (F) incorporation by mDCs performed on confocal microscopy micrographs using the software-assisted technique. Number of cells analyzed = 200. One representative experiment of 3 performed.