Evaluation of in vitro infection.

Murine macrophages (5×105 cells) were infected with stationary promastigotes of L. amazonensis (1×106 and 5×106, by a ratio of 1∶2 or 1∶10 macrophage per parasites, respectively) and the cultures were incubated for 24 h at 37°C, 5% CO2. Next, free parasites were removed and the percentage of infected cells and the number of amastigotes per macrophage in each passage (R0, R10, R20, and R30) were analyzed by counting 200 cells in triplicate. Mean ± SD is shown. Data shown are representative of three separate experiments, performed in triplicate, which presented similar results.