Figure 5.

NET release depends on amoeba viability. (A) Neutrophils (3 × 10⁵) were co-cultured with viable, formaldehyde-fixed or heat-inactivated trophozoites (1.5 × 10⁴) in RPMI medium for 4 h. PMA at 20 nM was used as positive control of NETosis. Supernatants were collected, mixed 1:1 with SYTOX Green (500 nM) and fluorescence was measured. NETs amount is expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments in triplicate. *p < 0.001. (B) Neutrophils were stimulated with 20 nM PMA or viable or non-viable amoebas (ratio 20:1) during 4 h; cells were fixed and staining with DAPI. Control neutrophils (i), control amoebas (ii), neutrophils treated with PMA (iii), neutrophils co-cultured with viable (iv), fixed (v), or heat-inactivated amoebas (vi). Images were taken at 60x magnification. Scale bar 50 μm.