2.3.2. Trypomastigote/Amastigote Susceptibility Assay

This assay was performed in 96-well microplates. Briefly, 4 × 10³ L-929 fibroblasts were seeded in 80 µl of MEM containing 10% FBS and antibiotics. The plate was placed overnight at 37 °C in a 5% CO₂ humidified incubator to allow cell adherence. Afterward, the cells were infected with 20 µL of trypomastigotes (40,000 trypomastigotes/well (4000 mammalian cells)) diluted in the same medium. The cell:parasite ratio was 1:10. Plates were incubated for 2 h at 33 °C and 5% CO₂. The medium was then discarded to dispose of nonpenetrated parasites and replaced with 200 µL of MEM without phenol red supplemented with FBS and antibiotics. After 48 h of incubation, the content of the wells was replaced with a fresh medium containing the dilutions of the compounds. The plates were returned to the incubator for an additional 96 h under the same conditions. Wells containing drug blanks, medium, fibroblasts, and untreated infected cells were included in all the plates as controls. An internal control of the reference drug (benznidazole) was also included. Next, 50 µl of substrate CPRG (diluted in 3% Triton X-100 solution) were added at a final concentration of 400 µM. After 18–20 h of incubation at 37 °C, absorbance readings were taken at 595 nm using a scanning multiwell spectrophotometer. All the assays were performed in triplicate and the experiments were run three times in different weeks. The results are expressed as the percentage of trypomastigote/amastigote growth inhibition (% T/A GI value) as follows:

where AE is the absorbance of the experimental group, AEB the absorbance of the compound blank, AC the absorbance of the control group, and ACB the absorbance of the culture medium blank. The IC₅₀ values (the drug concentration that eliminates 50% of the parasites) were determined for each compound using the GraphPad Prism software.