Evos fl cell imaging system m5000

After 24 h incubation of promastigotes of L. amazonensis and epimastigotes of T. cruzi at the IC₉₀ levels of the pure compounds, the cells were centrifuged at 1500 rpm at 4 °C for 10 min. Next, 50 µL of the pellet was deposited on a pre-coated coverslip and rested for 1 h. The parasites were fixed with formaldehyde (4%) for 30 min, washed in PBS 1X and treated with Triton (0.3%) for 10 min. For direct staining fluorescence, the cells were incubated with phalloidin–tetramethylrhodamine B isothiocyanate (phalloidin-TRITC; SigmaAldrich, Madrid, Spain) for 1 h at room temperature. Finally, the cells were washed with PBS 1X and a drop of mounting DAPI solution was added (4′,6-Diamidino-2-phenylindole dihydrochloride; Sigma-Aldrich; Madrid). The promastigotes and epimastigotes of the parasites tested were examined by Z-stack imaging using an EVOS™ FL Cell Imaging System M5000 (Life Technologies, EE. UU.) at λ_exc = 540 nm and λ_em = 570 nm.