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. 2020 Feb 24;9(2):508. doi: 10.3390/cells9020508

Figure 3.

Figure 3

The adopted CNN model, contrary to DNA release measurement, confirmed the inhibitory effect of NEi on NETs release. Peripheral blood neutrophils were isolated from peripheral blood of six healthy donors, samples were pretreated with NEi for 30 min and/or stimulated with peroxynitrite for 2 h. Negative control—Unstimulated cells, incubated only with RPMI 1640. (a) Representative images of samples pretreated with NEi and/or stimulated with peroxynitrite using live imaging with Hoechst 33342 and SYTOX Green; (b) representative images of immunofluorescently-labeled samples pretreated with NEi and/or stimulated with peroxynitrite; (c, d) for each patient, 10 images of unfixed cells stained with Hoechst 33342 and SYTOX Green were taken per each one of four experimental conditions (negative control; cells pretreated with NEi; cells stimulated with peroxynitrite; cells pretreated with NEi and stimulated with peroxynitrite). The images were analyzed using the adopted model and the results were analyzed as percentage of objects of different classes and compared between groups. (d) n = 6, 2-way ANOVA with post-hoc Tukey’s test. (e) At the indicated timepoint, NETs formation was assessed fluorometrically using SYTOX green after detachment of DNA with MNase, n = 6, 1-way ANOVA with posthoc Bonferroni’s test.