Spectramax 190 microplate spectrofluorometer

Log-phase promastigotes and one-day axenic amastigotes forms, of each isolate, were tested against Sb^V and Sb^III to measure the half-maximal inhibitory concentration (IC₅₀) induced by each drug. The IC₅₀ was determined by AlamarBlue reduction assay as previously described^{59 (link)}, with some modifications. Briefly, each parasite form was seeded in 96-well plates in triplicate at adequate conditions: log-phase promastigotes (4 × 10⁶ parasites/mL) and one-day axenic amastigotes (5 × 10⁵ parasites/mL) in 0.1 mL of Schneider medium (pH 7.2 for promastigotes or pH 5.5 for axenic amastigotes) supplemented with 20% of FBS and each drug in decreasing concentrations, leaving one column without any drug to serve as the control. Sb^V concentrations ranged from 20 mg/mL to 6 × 10^–4 mg/mL; Sb^III concentrations ranged from 0.196 mg/mL to 5 × 10^–6 mg/mL, with a 2:1 dilution factor between each one. After parasites incubation (Promastigotes: 26 °C, 48 h; Axenic amastigotes: 32 °C, 24 h), AlamarBlue reagent was added to each well (10 µL) followed by a new incubation at their respective temperatures for 4 h. Then, each plate was read on a Spectramax 190 microplate spectrofluorometer (Molecular Devices Corporation) at 570 excitation and 590 nm emission wavelengths, and the percentage of reduction of AlamarBlue was determined.