Dmem medium

All compounds were subjected to the MTT colorimetric assay using Balb-c 3T3 clone A31 cells (mouse fibroblast cells) acquired from the Rio de Janeiro Cell Bank (BCRJ code 0047). Inhibition values were determined in the cell-based assay as previously described [46 (link)]. Briefly, cells were cultured at 37°C in an atmosphere of 5% CO₂ using DMEM medium (Cultilab, Campinas-SP, Brazil) supplemented with 3.5 g glucose (Sigma-Aldrich), 1% penicillin/streptomycin solution and 10% FBS (Cultilab). Cells were plated at a concentration of 10⁵ cell/mL in 96-well plates and incubated for 24 h. All compounds were freshly diluted from 50 mM DMSO stock solutions to obtain the final concentration of 250 μM and added to each well by replacing the medium. The cell viability was assessed during 24, 48 and 72 h using the MTT (Sigma-Aldrich) reagent, with an incubation time of 3 h. Formazan crystals were dissolved using a solubility reagent composed of DMSO, glacial acetic acid and extran for 1 h. The readout was obtained using Biotek Synergy HT plate reader at 570 nm. Benznidazole was used as control. This assay was done in quatruplicate in two independent experiments. Statistical analyses were made in GraphPad Prism 5. Dunnett’s multiple comparison tests were performed using benznidazole as the standard compound for the ordinary ANOVA analysis using 95% confidence interval.

VERO cells were cultivated in 24-well plates (Nest, USA) in DMEM medium (Cultilab Ltd.) containing 10% FBS, for 48 h at 37ºC, in a 5% CO₂ atmosphere. The isolated strains were cultivated in LB broth for 18 h at 37ºC. An aliquot was centrifuged at 13,000 g for 2 min and the supernatant was filtered through 0.22 µm membrane filters (Millipore). Bacterial culture and supernatant filtrates aliquots were also boiled for 10 min. Fifty microliters of bacterial culture, supernatant, boiled bacterial culture or boiled supernatant were added to VERO cells, and after incubation for 18 h at 37ºC, the cells were washed three times with PBS, fixed with methanol and stained using May-Grünwald and Giemsa solutions. The results were compared with control cells. Cytotoxicity was considered positive when damaged cells were observed.

The B16F10 murine melanoma cell line was purchased from the American Type Culture Collection (Manassas, USA). The cells were grown in 75 cm² flasks with DMEM medium (Cultilab, Brazil) supplemented with 10% heat-inactivated fetal bovine serum (Cultilab, Brazil), 2 mM L-glutamine (Sigma Chemical Company, USA) and 0.1 g/mL streptomycin (FontouraWyeth AS, USA) at 37°C in a 5% CO₂ atmosphere.