T. brucei viability assay.

To determine the efficacy of inhibitors on trypanosome viability, 5 × 10³BSF cells were seeded into 96-well clear-bottom plates in 200 μL HMI-9 media, supplemented with compound or an equal volume of vehicle followed by incubation at 37°C at 5% CO₂ for three days. The Cell Titer Blue reagent (Promega; Madison, WI) was added (20 μL) to each well and the plates were incubated at 37°C at 5% CO₂ for three hours. Fluorescence at 585 nm was measured at 546 nm excitation using a GENios plate reader (Phoenix Research Products; Hayward, CA).