Vero cell monolayers were infected with trypomastigote forms of T. cruzi Tulahuen strain for 3 h and then washed and maintained in RPMI (Gibco Invitrogen Corporation) at 37°C in a 5% CO2 atmosphere. After 7 days, the parasites were collected from the supernatant of infected cells and harvested by centrifugation at 4,400 rpm for 5 min.
Peripheral blood mononuclear cells from non-chagasic donors were cultured with T. cruzi Tulahuen trypomastigotes (1:1 rate) for 3 h, then the cells were washed and cultured with recombinant bioactive IL-6 (20 ng/mL) or with anti-IL-6 (2 µg/mL) (Biolegend) or anti-IL-6 plus anti-IL-1β (2 µg/mL), or maintained in medium alone for 48 h. Then, culture supernatants were evaluated for IL-1β/NO levels, and the cells were analyzed for NT staining or NO production by flow cytometry. In addition, peripheral blood from chagasic patients and control donors (250 µL) were cultured with 7,500 trypomastigotes with or without IL-6 (20 ng/mL) or maintained in medium for 24 h and then cells were harvested to evaluate NO and ROS production and NT by flow cytometry.
Peripheral blood mononuclear cells from non-chagasic donors were cultured with T. cruzi Tulahuen trypomastigotes (1:1 rate) for 3 h, then the cells were washed and cultured with recombinant bioactive IL-6 (20 ng/mL) or with anti-IL-6 (2 µg/mL) (Biolegend) or anti-IL-6 plus anti-IL-1β (2 µg/mL), or maintained in medium alone for 48 h. Then, culture supernatants were evaluated for IL-1β/NO levels, and the cells were analyzed for NT staining or NO production by flow cytometry. In addition, peripheral blood from chagasic patients and control donors (250 µL) were cultured with 7,500 trypomastigotes with or without IL-6 (20 ng/mL) or maintained in medium for 24 h and then cells were harvested to evaluate NO and ROS production and NT by flow cytometry.