Mitochondrial Membrane Potential in L. amazonensis

For the determination of the mitochondrial membrane potential in the promastigote forms of L. amazonensis, we used tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes, Carlsbad, CA, USA) and flow cytometry. For this method, 2 × 10⁶ parasites/mL were treated with (-)-5-demethoxygrandisin B IC₅₀ for 24 h at 26 °C. Nontreated parasites were used as a negative control, and heat-killed parasites (60 °C bath for 30 min) were used as a positive control. Post-incubation, the parasites were subjected to centrifugation at 1500× g for 5 min at room temperature. Afterward, they were washed with PBS and incubated in a solution of 300 μL TMRE (50 nM) in the absence of light for 15 min at room temperature. Subsequently, a flow cytometry analysis was conducted using a CytoFlex flow cytometer (Beckman Coulter Life Sciences, Inc., Brea, CA, USA). TMRE excitation was achieved using a 488 nm blue laser, and the emitted fluorescence was captured with a 585/42 nm bandpass filter. The flow cytometry data were analyzed using CytExpert software version 2.1 (Beckman Coulter Life Sciences, Inc., Brea, CA, USA) [26 (link)].