*2.3. NET Quantitation Assay*

NET quantitation was performed as described before [43]. In brief, neutrophils (5 × <sup>10</sup>5) were centrifuged at 4000 rpm for 2 min and resuspended in 500 <sup>μ</sup>L of RPMI-1640 medium (Biological Industries) supplemented with 5% fetal bovine serum (FBS, Gibco) and 500 nM SYTOX® Green (Invitrogen). A volume of 100 <sup>μ</sup>L of cell suspension (1 × <sup>10</sup><sup>5</sup> neutrophils) was added to a 96 well plate, allowed to sediment for 20 min at 37 ◦C and then, stimulated with 1 × <sup>10</sup>3, 2 × <sup>10</sup>3, 5 × 103 or 1 × <sup>10</sup><sup>4</sup> *E. histolytica* viable trophozoites (trophozoite:neutrophil ratios 1:100, 1:50, 1:20 and 1:10, respectively). In other experiments, neutrophils were stimulated with 5 × <sup>10</sup><sup>3</sup> formaldehyde-fixed or heat-inactivated trophozoites. Co-cultures were incubated at 37 ◦C and fluorescence was measured during 4 h from the well bottom using a spectrofluorometer Synergy HTX (BioTek) with 485 nm excitation and 528 nm emission filters. NETosis induced by PMA (50 nM, Merck) and A23187 (10 μM, Merck) were used as positive controls.

To determine the role of NADPH-oxidase in amoeba-induced NETosis, neutrophils (5 × <sup>10</sup>5) were resuspended in 500 <sup>μ</sup>L of PBS and pretreated with the inhibitor apocynin (400 μM) or vehicle DMSO (0.1%) for 30 min at 4 ◦C (all reagents were supplied by Merck). After pretreatment, neutrophils were induced to NETosis with trophozoites as described above. To determine the role of ROS from neutrophils, these cells (5 × 105) were resuspended in 500 μL of PBS and pretreated separately for 30 min at 4 ◦C with the ROS scavengers pyrocatechol (200 μM), catalase (200 UI/mL), luminol (50, 100 and 200 μM), isoluminol (50, 100 and 200 μM) or mitoTEMPO (400 μM). As negative controls, neutrophils were pretreated with the corresponding vehicles (all reagents were supplied by Merck). After pretreatments, neutrophils were immediately tested for amoeba-induced NETosis as described above in culture media added with the inhibitors or scavengers at concentrations indicated previously (except for mitoTEMPO). All experiments were performed three times in triplicates.
