Spectramax spectrofluorometer

The mitochondrial transmembrane electric potential (ΔΨm) was investigated using the JC-1 fluorochrome, which is a lipophilic cationic mitochondrial vital dye that exhibits accumulation in mitochondria in response to ΔΨm. The fluorochrome exists as a monomer at low concentrations, where the emission is 530 nm (green fluorescence), but at higher concentrations, it forms J-aggregates after accumulation in the mitochondria, where the emission is 590 nm (red fluorescence). To this, H. capsulatum yeasts (10⁶ viable cells), treated or not with mebendazole for 72 h were harvested, washed in PBS, and incubated in a 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. To evaluate the ΔΨm, 10⁶ fungal cells were incubated with 10 μg/mL JC-1 solution for 30 min with readings made every minute using a microplate reader (SpectraMax spectrofluorometer, Molecular Devices). As positive control of the depolarization of the mitochondrial membrane, fungal cells were incubated with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) at 1 μM, a mitochondrial protonophore. The relative ΔΨm value was obtained by calculating the ratio between the reading at 590 nm and the reading at 530 nm (590:530 ratio).

The general metabolism of the parasites was evaluated by a resazurin dye/Alamar blue (7-hydroxy-3H-phenoxazin-3-one-10-oxide) assay (Sigma-Aldrich, St Louis, MO, USA) [20 (link)]. L. amazonensis and L. chagasi promastigotes were incubated in sterile 96-well plates (in a total volume of 100 μL culture medium/well) and then resazurin was added to a final concentration of 0.0125% in PBS [21 (link)]. After a 4 h incubation at room temperature, parasites were analyzed in a microplate reader (SpectraMax spectrofluorometer, Molecular Devices, San Jose, CA, USA) using a pair of 590 and 544 nm as emission and excitation wavelengths, respectively. The viability was evaluated based on a comparison with untreated control cells. Parasites were also treated with sodium azide (40 µM) for 30 min in order to obtain nonviable cells to use as a positive control in the viability test.

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).