Dimethyl sulphoxide

Exponential-phase pXG-mCherry12 parasites were seeded in black 96-well plates with clear bottom (200 μL per well) with increasing concentrations of amphotericin B and miltefosine, diluted in M199 medium and maintained at 26°C. After 48 and 72 h of incubation, the half-maximal effective concentration (EC₅₀) was measured by two different techniques: fluorimetric and colorimetric methods. Fluorescence was monitored at a 570-nm excitation wavelength and a 620-nm emission wavelength using a BMG FLUOstar Optima microplate reader (BMG LabTech, Ortenberg, Germany). On the other hand, for the colorimetric assay 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) was used as previously described [19 (link)]. MTT solutions were prepared at 5 mg/mL in PBS. After adding 20 μL of MTT solution to each well, the plates were incubated during 4 h at 26°C. Subsequently, 80 μL of dimethyl sulphoxide (DMSO, Panreac, Spain) was added to each well to dissolve formazan crystals. The optical density (OD) was measured using a Multiskan EX Microplate Photometer plate reader at 540 nm.
The EC₅₀ was obtained by fitting a sigmoidal Emax model to dose-response curves. In both cases, promastigotes viability was evaluated based on a comparison with untreated control cells. The results were expressed as mean ± standard error (SEM) from three independent experiments.

In order to assess ATO cytotoxicity, an alamarBlue Assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed using serial dilutions after 72 h exposure to ATO (0.96 to 126.3 µM, Sigma-Aldrich, San Louis, MO, USA). In this case, 5-fluorouracil (0.23 to 38.4 µM, 5-FU, Sigma-Aldrich, San Louis, MO, USA) was used as a positive control. To prepare the aqueous solution (5 mg/mL) of compounds, ATO (5 mg) and 5-FU (5 mg) were dissolved in DMSO PA, 99% (DMSO) (1 mL) (Dimethyl sulphoxide, Panreac). Absorbance was measured at 570 nm (oxidized/blue) and 600 nm (reduced/pink) wavelengths using a SpectraMax 190 microplate reader (Molecular Devices, San Jose, CA, USA). The calculation was obtained using the formula ARLW (percent of reduced cells) = ALW (oxidized cells in 570 nm) − (AHW/reduced cells in 600 nm × RO) × 100, in which R0 (correction factor) = AOLW/AOHW (blank samples), according to the alamarBlue Assay (Thermo Fisher Scientific, Waltham, MA, USA). The percentage of inhibition of the tested drugs (Arsenic Trioxide and 5-FU) was calculated using the mean of cell viability of the negative group control (DMSO) as the reference group. For this assay, three independent experiments were carried out in duplicate.