The HMCs were plated onto gelatine-coated glass coverslips in 24-well plates (105 cells/well) and infected at a 10:1 parasite:host cell ratio after 24 h. Afterwards EPZ-6438 price the cultures were washed, and the NQs (0.5

to 20 μM) were added. At specified intervals, the cultures were fixed in Bouin’s solution, stained with Giemsa and counted to assess the following parameters: percentage of cells infected, number of parasites/infected cell and the endocytic index (EI), which refers to the number of parasites/100 cells [52]. The IC50 values for the different days of treatment, corresponding to the concentration that led to 50% inhibition of each parameter, were calculated. To determine the possible toxic selleck inhibitor effects of the compounds on the host cells, uninfected macrophages and HMCs were incubated at 37°C with the NQs. After 2 days, the viability of the cells was measured using the MTT colorimetric assay [53]. The absorbance was measured at 490 nm with a spectrophotometer (VERSAmax Tunable, Molecular Devices, USA), allowing for the determination of an LC50 value, which is the concentration that reduces cellular viability by 50%. Transmission and scanning electron microscopy analysis Epimastigotes (5×106 cells/mL)

were treated for 24 h with the selected NQs at their respective IC50/24 h values in LIT medium at 28°C. Afterward, they were fixed with 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) for 40 min at 25°C and post-fixed with 1% OsO4, 0.8% potassium ferricyanide and 2.5 mM CaCl2 in the same buffer for 20 min at 25°C. The cells were dehydrated in an ascending acetone series and embedded in PolyBed 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Jeol JEM1011

transmission electron microscope (Tokyo, Japan). Alternatively, dehydrated samples were dried by the critical point method with CO2, mounted on aluminum stubs, coated with a 20 nm thick gold layer and examined on a Jeol JSM6390LV scanning electron microscope (Tokyo, Japan). Both electron microscopes Phospholipase D1 are located in Plataforma de Microscopia Eletrônica at Instituto Oswaldo Cruz (Fiocruz). Flow cytometry analysis Epimastigotes were treated for 24 h with the NQs at concentrations up to their IC50 values. We then determined the mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) production. For ΔΨm analysis, the parasites were incubated with 50 nM tetramethylrhodamine (TMRE) (Molecular Combretastatin A4 order Probes, Carlsbad, USA) for 15 min at 28°C, using 10 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich Chemical Co.) as a control for ΔΨm dissipation. Alterations in TMRE fluorescence were quantified using an index of variation (IV), which was calculated using the equation (MT – MC)/MC, where MT is the median of fluorescence for treated parasites and MC is the median of fluorescence of the control parasites.