*3.1. E. histolytica Trophozoites Induce NETosis in a Dose-Dependent Manner*

Herein, we evaluate amoeba-induced NETosis under different amoeba:neutrophil ratios. As shown in Figure 1A, amoebas trigger NET release in a dose-dependent manner at ratios between 1:100 to 1:20. The maximum level of DNA release was obtained at ratio 1:20, as no significant differences were observed with respect to the 1:10 ratio and the positive controls PMA and A23187.

NET release was confirmed by detecting MPO in the DNA by immunofluorescence. As shown in Figure 1B, untreated control neutrophils have condensed, multilobed nucleus and MPO located in the cytoplasmic compartment as expected. The positive controls PMA and A23187 induced nuclear decondensation and formation of DNA fibers that were colocalized with MPO. On the other hand, *E. histolytica* trophozoites induced cloudy NETs with heterogenous distribution of MPO. To ensure that these structures correspond to NET, we observed that acetylated histone H4 and NE also colocalized with released DNA in response to trophozoites (Figure 1C).
