2.7. Plasmatic membrane permeability assay

To determine the alterations in the plasmatic membrane permeability the SYTOX® Green nucleic acid stain fluorescent dye (ThermoFisher Scientific, MA, USA) was used. This probe is normally impermeable to intact plasma membranes, but is able to penetrate cells with increased permeability, reaching the nucleus and binding to nucleic acids, resulting in a strong increase in its fluorescence. The epimastigotes were incubated with the IC₉₀ of the compounds for 24 h at 26 °C. Then, the solution was centrifuged (825 g, 10 min, 4 °C) and resuspended in 50 μl of buffer with 1 μM concentration of the kit. The measure of green cells concentration was developed using the Countess II FL (ThermoFisher Scientific, Madrid, Spain) and the images were done using GFP light cube in the EVOS® FL Cell Imaging System (ThermoFisher Scientific, Madrid, Spain). A positive control (Triton 0.5% for 30 min), a reference treatment (benznidazole) and a negative control (without any treatment) were added (López-Arencibia et al., 2019b).