2.6. Macrophage-Amastigote Model

We used a previously described methodology [39], with some modifications. Macrophage monolayers, grown in RMPIc medium, for 3 days, were detached from the bottom of the culture chamber, by physical treatment (low temperature and light strokes). Obtained macrophages were seeded in flat-bottom 96-well culture plates, at a density of 2 × 10⁵ cells mL⁻¹ (40,000 macrophages/well), in a final volume of 200 µL. Plates were incubated for 2 h, and the supernatant was discarded to remove non-adhered macrophages. Medium was replaced with stationary-phase promastigotes suspended in 200 µL of RPMIc (2 × 10⁶ parasites mL⁻¹, 400,000 parasites/well), at a macrophage: parasite ratio 1:10 [40]. The plates were incubated for 4 h. Afterwards the medium was replaced with fresh RPMIc and increasing concentrations of compounds or drugs tested were applied, in volumes of 2 µL, according to the different treatment groups. Plates were kept for 72 h, at 37 °C and 5% CO₂. The medium was discarded and the wells were washed twice with PBS. Next, a Giemsa staining was performed in situ. It consisted of adding 200 µL of methanol/well, incubating for 5 min, discarding the fixative, and letting it dry at room temperature. Later, the Giemsa solution (10% in PBS w/v, 200 µL/well) was added, incubated for 10 min, and washed with PBS, allowing it to dry. Finally, the bottom of the plates was detached by percussion and the circular bottoms (wells) were fixed to slides with Permount synthetic resin, for later evaluation (Alcazar W., manuscript in preparation). Cell counting was performed with a 100× immersion objective. A minimum of 200 random macrophages were examined. The percentage of infected macrophages (% infection) and the average number of amastigotes present in infected macrophages (_amastigotes) were determined. From these data, the infection index (inf-I) was calculated: inf-I = % infection × _amastigotes. The infection rate represents the number of intracellular amastigotes per 100 macrophages evaluated [40,41]. Additionally, the GI₅₀ for the decrease in the infection rate was calculated. At least three independent experiments were performed. The effect of amphotericin-B (AmB) [Sigma-Aldrich, (St. Louis, MO, USA)] was included as positive control of the assay.