2.2.3. BNZ activity against extracellular trypomastigote forms 
BNZ activity against culture-derived trypomastigote forms was performed by seeding 2 ×106 trypomastigotes·mL–1, and after the addition of BNZ at a concentration range from 100 to 0.4 μM (200 μL·well–1 volumes) in 96-well plates at 37º C in humidified 95 % air, 5 % CO2 atmosphere for 24 h. Untreated controls were also included. 20 μL of resazurin sodium salt (0.125 mg·mL–1) (Sigma-Aldrich) was added, and the plates were incubated for further 4 h (Martín-Escolano et al. , 2019). Finally, trypanocidal activity was determined following the same procedure as described to assess the trypanocidal activity against epimastigote forms. Each compound concentration was tested in triplicate in four separate determinations.
Since the half-life of culture-derived trypomastigotes (without host cells) was determined to be 48 h, the assay was restricted to 24 h of treatment.