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. 2019 Feb 4;9:1283. doi: 10.1038/s41598-018-37852-5

Figure 4.

Figure 4

SLE- IgG increased neutrophil NETosis in the presence of endothelial cells. (A) PMA-induced NETosis was confirmed by immunofluorescence. Neutrophils were incubated on coverslips in the absence or presence of 40 nM PMA and then fixed. Coverslips were then stained for histone H3 and mounted using a DAPI-mounting medium. (B) Cell supernatants from neutrophils cultured in the absence or presence of 40 nM were analysed using the Quanti-iT PicoGreen dsDNA kit. Data are presented as the mean and SEM of three independent experiments. Statistical significance was determined by a Mann-Whitney test. (C–E) IgG-mediated extracellular DNA release was assessed in (C) the absence of an endothelial monolayer; (D) the presence of resting endothelial cells; and (E) the presence of activated endothelial cells. Neutrophils were cultured for 4 hours in the absence or presence of 40 nM PMA, after which cell supernatants were assessed for cell-free dsDNA. For endothelial cell activation, HUVEC were pre-stimulated with 10 ng/ml TNF-α for 24 hours and washed with warmed PBS prior to addition of neutrophils. Data are presented as the mean and SEM of three independent experiments and analysed using a two-way ANOVA with a Dunnet’s multiple comparison test. *p < 0.05, **p < 0.01, ****p < 0.0001.