Xtt tests

Cell toxicity of BLT-1 was evaluated by colorimetric XTT tests (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, BUVEC (n = 3) were cultured in 96-well plates (Greiner) and treated with BLT-1 (2 µM) in a total volume of 50 µL for 72 h. Thereafter, 50 μL of the XTT working solution were added and the samples were incubated for 4 h (37 °C, 5% CO₂ atmosphere). The resulting formazan products were estimated via optical density (OD) measurements at 590 nm and reference filter 620 nm wavelength using a Varioskan^® Flash Multimode Reader (Thermo Scientific). BUVEC treated with a solvent (DMSO; 0.02%) were used as the negative control.
For experiments on parasite viability, 5 × 10⁵ tachyzoites/sporozoites of each parasite species were treated for 2 h with BLT-1 (2 µM; 37 °C, 5% CO₂). The viability was determined using the trypan blue (Sigma-Aldrich) exclusion test. Non-stained parasites were considered to be viable, as reported elsewhere [40 (link)].

Cell toxicity of P-gp inhibitors was assessed by colorimetric XTT tests (Promega, Madison, WI, USA) according to the manufacturer instructions. Briefly, BUVEC (n = 3) were cultured in 96-well plates (Greiner) and treated with verapamil (40 µM), valspodar (5 µM) or tariquidar (2 µM) in a total volume of 50 µl for 96 h. Thereafter, 50 μL of XTT working solution were added and the samples were incubated for 4 h (37 °C, 5% CO₂ atmosphere). The resulting formazan products were estimated via optical density (OD) measurements at 590 nm and reference filter 620-nm wavelength using VarioskanTM Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). BUVEC treated with the solvent (DMSO; 0.01%) were used as negative controls.
Additionally, for experiments on parasite viability, 5 × 10⁵ tachyzoites of each parasite species were treated for 1 h with each of the studied compounds (verapamil 40 µM, valspodar 5 µM and tariquidar 2 µM; 37 °C, 5% CO₂). Viability of tachyzoites was determined by the trypan blue (Sigma-Aldrich) exclusion staining assay [60 (link)]. Non-stained parasites were considered as viable.