*2.6. Mitochondrial ROS Quantitation in Neutrophils*

Isolated neutrophils (5 × <sup>10</sup>5) were resuspended in 500 <sup>μ</sup>L of PBS added with MitoSOXTM Red (10 μM) and incubated for 30 min at 4 ◦C. Cells were centrifuged at 4000 rpm for 2 min and resuspended in 500 μL of RPMI-1640 medium supplemented with 5% FBS. Each 100 <sup>μ</sup>L of MitoSOX pretreated neutrophils (1 × <sup>10</sup>4) were transferred to 96 well plate and stimulated with 1 × 103, 2 × 103, 5 × <sup>10</sup><sup>3</sup> or 1 × <sup>10</sup><sup>4</sup> viable amoebas (trophozoite:neutrophil ratios 1:100, 1:50, 1:20 and 1:10). Fluorescence was read from the well's bottom after 2 h using a spectrofluorometer Synergy HTX (BioTek) with 485 nm excitation and 580 nm emission filters. PMA (50 nM) and A23187 (10 μM) were used as negative and positive controls of mitochondrial ROS, respectively.
