Figure 1
NETs carry miRNA cargo. Neutrophils from healthy donors were exposed to four different activators, including PMA (100 nM), amyloid fibrils (A25T, 3 μM), IL-8 (50 ng/mL) and L. amazonensis promastigotes (La; ratio of 5 parasites/neutrophil) for 3 h. (A) Measurements obtained from DNA (PicoGreen dsDNA kit) and miRNA (Agilent 2100 RNA Bioanalyzer) for each stimulus. (B) Correlation between DNA and miRNA quantities from A. (C) Quantitative PCR results for the hsa-miR-142-3p Taqman assay displaying the mean expression levels ± SEM of the test groups (La and A25T or IL-8) in relation to the control (PMA) in donor-paired analysis from 6 independent experiments for each activator. Statistical significance was calculated by a two-tailed test (**p < 0.001; *p < 0.05). (D) Live-cell imaging of NET formation in two different channels for up to 120 minutes. SYTOX Green at 10 nM was used for NET detection (depicted in green; left panels), and a TYE™-665 labeled hsa-miR142-3p locked nucleic acid (LNA) detection probe at 5 μM for miR-142-3p detection (depicted in red; middle panels). The hsa-miR-142-3p staining was present in two different morphological patterns: one shows a strong staining (asterisk), and the other reveals a weak staining that can be either punctate (blue arrow) or diffuse (white arrows). The right panels show the merge of the two channels. Bars: 20 μm.
