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.