Infection of H9c2 cells by T. cruzi

H9c2 cells were seeded in T-225 flasks (5 × 10⁶ cells/flask, 225 cm² culture surface; Corning Inc., NY, USA) in DMEM-10% FBS for 4 h to allow attachment. Cells were then washed once with PBS before infection. T. cruzi trypomastigotes, collected at days 5 to 8 after infection, from LLC-MK2 parasite infected cultures, were allowed to swim out for 4 hours at 37°C from a centrifuged pellet (2,500 rpm/10 min/RT; [15 (link)]). Trypomastigotes were then collected and counted in a CASY Cell Counter (Roche-Applied-Science) using the 60 μm capillar. Trypomastigotes, in supplemented assay DMEM, were added to H9c2 cultures in a multiplicity of infection (MOI) = 1 and incubated for 18 hours. Cells were washed once with PBS before incubation of the infected H9c2 monolayer with trypsin (Life-Technologies) to detach cells from the flask. Cells were counted in a CASY Cell Counter (Roche-Applied-Science) using the 150 μm capillar and their density set at 5×10⁴ cells per ml in supplemented assay DMEM.