Mitochondrial Membrane Potential Measurement

Mitochondrial membrane potential (ΔΨm) was investigated using the JC-1 fluorochrome, as described by Macedo-Silva et al. (2011 (link)). Briefly, control an BEL-treated promastigotes (2.5 μM, 1 h) were harvested, washed in PBS pH 7.2, added to the reaction medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES/K⁺ pH 7.2, 2 mM Pi, 1 mM MgCl₂ and 500 μM EGTA and counted using a hemocytometer. To evaluate the ΔΨm, 2.0 × 10⁷ parasites were incubated with 10 μg/mL JC-1 for 25 min in the reaction medium, and readings were made every min immediately after the start of the 25-min incubation period, using a Molecular Devices Microplate Reader (SpectraMax spectrofluorometer). As a positive control for mitochondrial membrane depolarization, parasites were incubated with 2 μM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP). The relative ΔΨm value was obtained by calculating the ratio 590 nm (red)/530 nm (green).