Figure 2
miR-142-3p staining pattern throughout NET formation steps. Neutrophils were activated with fixed L. amazonensis promastigotes (La; ratio of 5 parasites/neutrophil) for up to 120 min, and directly monitored in vitro by live-cell imaging using differential interference contrast (DIC), SYTOX Green as a NET marker and LNA-miR-142-3p-TYE™665 as a miRNA marker. (A,B) The morphological characteristics of NET formation are represented, depicting the loss of the classical neutrophil lobulated nuclei with decondensed chromatin, as well as hsa-miR-142-3p staining (red) as a diffuse, punctate pattern, in the neutrophil cytoplasm (1). A cell with the nuclear membrane starting to disintegrate (2) and another with amorphous nuclear material filling most of the cell (3) can be seen. The images also show the extrusion of a diffuse NET (4), and a spread-out NET with the DNA scaffold forming a web-like structure, with hsa-miR-142-3p staining (red) colocalized with DNA (green) (5). Two NET-trapped Leishmania promastigotes (Fig. A, arrows) and non-stained viable neutrophils were observed. (C) LNA-miR-142-3p-TYE™665 (5 μM) detection (red). (D) NET staining by SYTOX Green (10 nM). Bars: 25 μm.
