*2.8. ROS Scavenging from Amoebic Trophozoites*

Trophozoites (5 × <sup>10</sup>5) were resuspended in 500 <sup>μ</sup>L of PBS added with the ROS scavengers pyrocatechol (50, 100 or 200 μM) or luminol (50, 100 or 200 μM), as well as with the vehicle DMSO (0.1%), and incubated during 30 min at 37 ◦C. Then, the cell suspension was added with H2DCFDA (100 μM) and ROS quantitation determined as mentioned above. On the other hand, to determine the role of amoebas-derived ROS in the NETosis process, trophozoites (5 × <sup>10</sup>5) were resuspended in 500 <sup>μ</sup>L of PBS added with the ROS scavengers pyrocatechol (50, 100 and 200 μM) or luminol (50, 100 and 200 μM). Trophozoites were incubated for 1.5 h at 37 ◦C, centrifuged at 4000 rpm for 2 min and then resuspended in 500 μL of PBS to be used immediately for NET induction.

#### *2.9. Visualization of Amoebas-Derived ROS*

Trophozoites treated for ROS quantitation as well as trophozoites treated for ROS depletion with the scavenger pyrocatechol as mentioned above, were fixed with formaldehyde (3.5%) and counterstained with DAPI (5 μg/mL). An aliquot of 20 μL was observed under fluorescence microscope Olympus BX51. Images were processed using ImageJ software.

#### *2.10. Detection of MPO Activity*

Neutrophils (5 × <sup>10</sup>5) were centrifuged at 4000 rpm for 2 min and resuspended in 500 μL of RPMI-1640 medium supplemented with 5% FBS (Gibco) and luminol (200 μM) or isoluminol (200 <sup>μ</sup>M). Each 100 <sup>μ</sup>L of cell suspension (1 × <sup>10</sup><sup>5</sup> neutrophils) was placed in a 96 well plate and cells were incubated for 20 min at 37 ◦C for sedimentation. Posteriorly, neutrophils were stimulated with 1 × <sup>10</sup>3, 2 × 103, 5 × 103 or 1 × <sup>10</sup><sup>4</sup> *E. histolytica* viable trophozoites. Co-cultures were incubated at 37 ◦C for 4 h and luminescence was measured every 5 min from the well bottom using a spectrofluorometer Synergy HTX. PMA (50 nM, Merck) and A23187 (10 μM, Merck) were used as controls.
