Fig. 2.

Capacity of Stx2 to induce NETosis in PMN from HC. a PMN (600,000/300 μl) from HC were incubated with medium (basal), PMA, MSU, or Stx2 for 4 h. Then, NET formation was revealed by staining DNA with PI, and elastase (a) or MPO (b) with specific antibodies. The images, representatives of 1 experiment of 8 performed with PMN isolated from healthy donors, were acquired via CLSM. Scale bars = 10 μm. c Quantification of the percentage of NETotic cells analyzed by CLSM. Viable cells and NETotic cells were counted in 5 photographs (50 cells) from each treatment. Then, the NET percentage was calculated as the number of NET over the number of total cells. Data are presented as the mean percentage ± SEM of cells evidencing characteristics of NETotic cells (decondensed chromatin and disintegrated nuclei) in which DNA colocalized with elastase, over the total number of cells. At least 50 cells per condition were counted in each experiment (n = 5). * p < 0.05 (Kruskal-Wallis test followed by Dunn's post hoc test). Extracellular DNA concentration (d) and elastase activity (e) in supernatants of the culture of PMN (4 × 10⁵ cells/ml) isolated from healthy donors incubated with medium (basal), Stx2, Stx2i, or Stx2ΔAB (0.1 μg/ml) for 4 h. Data are expressed as the mean ± SEM of 5 independent experiments. * p < 0.05 (Kruskal-Wallis test followed by Dunn's post hoc test).