*2.5. Intracellular ROS Quantitation in Neutrophils*

Neutrophils (5 × <sup>10</sup>5) were resuspended in 500 <sup>μ</sup>L of PBS added with 10 <sup>μ</sup>M 2- ,7- dichlorofluorescein diacetate (H2DCFDA) and incubated for 30 min at 37 ◦C in the dark. Cells were centrifuged at 4000 rpm for 2 min and resuspended in 500 μL of RPMI-1640 supplemented with 5% FBS. Subsequently, each 100 <sup>μ</sup>L of suspension (1 × <sup>10</sup><sup>5</sup> neutrophils) was transferred to 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><sup>3</sup> or 5 × <sup>10</sup><sup>3</sup> viable *E. histolytica* trophozoites (trophozoite:neutrophil ratios 1:100, 1:50 and 1:20). Fluorescence intensity was measured after incubation during 1 h at 37 ◦C from the well bottom in the spectrofluorometer Synergy HTX using 485 nm excitation and 528 nm emission filters. PMA (50 nM) and A23187 (10 μM) were used as positive controls for ROS production.
