*3.3. Dead Trophozoites Do Not Induce NET Release and Contain Scarce ROS*

As shown in Figure 3A, heat-killed or fixed trophozoites did not induce NETosis on human neutrophils, in contrast to viable amoebas that induced NET levels like PMA and A23187. This result suggests that metabolically active trophozoites are required for triggering NETosis. Since most studies suggest that NETosis requires a source of ROS, which is not related to neutrophils in this case, we explored the possibility that ROS from trophozoites was involved. So, we decided to quantify ROS in living and dead amoebas and found, as expected, that formaldehyde-fixed and heat-killed trophozoites exhibited far fewer ROS than live amoebas (Figure 3B).

**Figure 3.** Dead *E. histolytica* trophozoites do not trigger NETosis and possess less ROS. (**A**) Neutrophils (1 <sup>×</sup> <sup>10</sup>5) were culture in RPMI-1640 medium supplemented with 5% FBS and added with 500 nM SYTOX® Green. Cells were stimulated with PMA (50 nM), A23187 (10 <sup>μ</sup>M), viable trophozoites (5 <sup>×</sup> <sup>10</sup>3), heat-killed trophozoites (5 <sup>×</sup> 103) or formaldehyde-fixed trophozoites (5 <sup>×</sup> <sup>10</sup>3). Fluorescence was read at 4 h. (**B**) Viable, heat-killed and formaldehyde-fixed trophozoites were pretreated with H2DCFDA for 1 h. Posteriorly, amoebas (1 <sup>×</sup> 105) were placed in a 96 well plate and fluorescence was read. NET and ROS amounts are expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments. \* *p* < 0.0001.
