3.4. In Vitro Antitrypanosomal Assay

Trypanosoma brucei brucei 427 trypomastigotes were cultured in Iscove’s Modified Dulbecco’s medium (IMDM, Lonza, Basel Switzerland) and supplemented with 10% fetal calf serum, HMI-9 supplement [54], hypoxanthine and penicillin/streptomycin at 37 °C in a 5% CO₂ incubator. Serial dilutions of test compounds were incubated with the parasites in 96-well plates for 24 h, and residual parasite viability in the wells determined by adding 20 µL resazurin toxicology reagent (Sigma-Aldrich) and incubating for an additional 24 h. Reduction of resazurin to resorufin by viable parasites was assessed by fluorescence readings (excitation 560 nm, emission 590 nm) in a Spectramax M3 plate reader (Molecular Devices, San Jose, CA, USA). Fluorescence readings were converted to % parasite viability relative to the average readings obtained from untreated control wells. IC₅₀ values were determined by plotting % viability vs. log[compound] and performing non-linear regression using GraphPad Prism (v. 5.02) software [55].