*2.7. ROS Quantitation in E. histolytica Trophozoites*

Viable, formaldehyde-fixed or heat-inactivated trophozoites (5 × <sup>10</sup>5) were resuspended in 500 μL of PBS added with H2DCFDA (100 μM) and incubated for 1 h at 37 ◦C. After incubation, amoebas were centrifuged at 4000 rpm for 2 min and resuspended in 500 μL of RPMI-1640 medium supplemented with 5% FBS. Each 100 μL of the cell suspension (1 × <sup>10</sup><sup>5</sup> H2DCFDA-pretreated trophozoites) was added to 96 well plate and allowed us to sediment for 10 min at 37 ◦C. Fluorescence was read from well bottom using a spectrofluorometer Synergy HTX with 485 nm excitation and 528 nm emission filters.
