*2.4. NET Visualization*

NET immunofluorescence was performed as described previously [41] with some modifications. In brief, neutrophils (2 × <sup>10</sup>5) were resuspended in 100 <sup>μ</sup>L of RPMI-1640 medium supplemented with 5% FBS and seeded on coverslips pretreated with poly-L-lysine solution (Merck). After sedimentation for 20 min at room temperature, the neutrophils were stimulated with 1 × 104 viable trophozoites or positive controls PMA (50 nM) and A23187 (10 μM). Co-cultures were incubated for 4 h at 37 ◦C and then fixed with 3.7% formaldehyde during 10 min. Fixed cells were permeabilized using 0.2% Triton X-100 (BioRad) in PBS for 5 min. Detergent was washed out two times with cold PBS and blocking was carried out with a 1% BSA, 0.3 M glycine and 0.1% Tween 20 in PBS, for 30 min at 37 ◦C. Samples were then incubated with primary antibodies against NETs constituents: anti-NE (Santa Cruz Biotechnology, sc-365950), anti-MPO (Abcam, ab16886) or anti-acetylated histone H4 (Abcam, ab61238) antibodies diluted 1:100 in 1% BSA, 0.1% Tween 20 in PBS for 1 h at room temperature. Samples were washed two times with cold PBS and then incubated with secondary anti-mouse IgG-FITC (Merck, F5387) or anti-rabbit IgG-TRITC (Zymax) antibodies diluted 1:50 in the same solution as primary antibodies for 1 h at room temperature in the dark. After two washes with cold PBS, samples were stained with 5 μg/mL DAPI (Merck) and the coverslips were mounted on slides using Fluoroshield (Merk) before observation in a fluorescence microscope (Olympus BX51). Images were processed using ImageJ software.

For detection of MPO on the surface of amoebic trophozoites, immunofluorescence was performed as above but cell cultures were fixed for 5 and 10 min after the addition of parasites.
