Anthracyclines reduce baseline NETosis in a dose-dependent manner. NETosis assays were performed on human neutrophils resuspended with five concentrations of epirubicin, daunorubicin, doxorubicin, or idarubicin (0.0, 0.5, 1.0, 5.0, and 10.0 μM) and 5 μM Sytox Green fluorescence dye. Neutrophils were unstimulated in order to obtain baseline NETosis information. Fluorescence intensities (proxy for DNA release) were measured by a plate reader every 30 min for 4 h. % DNA release for each condition compared to Triton X-100 sample (100% DNA release) was calculated. Baseline NETosis by (A) epirubicin, (B) daunorubicin, (C) doxorubicin, and (D) idarubicin was reduced in a dose-dependent manner (n = 3, *
p < 0.05 between 10.0, 5.0 and 0.0 μM dosage; Two-way ANOVA with Bonferroni’s multiple comparison post-test). (E) Confocal microscopy of neutrophils (unstimulated and LPS-treated) were also performed on 5.0 μM samples of each anthracycline. Neutrophils were stained for DNA (blue) and myeloperoxidase (MPO) (green); colocalization of these stains indicates little NET release in each condition (n = 3; scale bar, 20 μm).