Parasite viability was then determined using the resazurin assay, which showed a great decrease in resazurin reduction after MDL28170 treatment, mainly with the two times the IC50 dose of the drug, when compared to non-treated control cells. This result indicated that the calpain CPI-613 inhibitor Crizotinib induced loss of parasite viability in a concentration-dependent manner. Incubation with JC-1 showed that cells treated with the calpain inhibitor at two times the IC50 dose had a significant reduction of Dym when compared with the control parasites. In addition, pre-incubation with FCCP resulted in decreased mitochondrial staining with JC-1. After 34 min of JC-1 uptake, the addition of 2 mM FCCP fully collapsed the Dym, including control parasites. Since loss of mitochondrial membrane potential was mainly observed in cells treated with two times the IC50 dose, this may be considered a secondary effect of MDL28170 treatment. In transmission electron microscopy, the results showed ultrastructural changes after 72 h of incubation with the inhibitor at IC50 and two times the IC50 doses in comparison to non-treated parasites. MDL28170treated cells showed an intense vacuolization in the cytoplasm. In addition, the calpain inhibitor was also able to induce an intense disorganization of the endocytic pathway, which is distended and presented reduced electron density as well as the accumulation of small vesicles characteristic of the multivesicular body network. The mitochondrion displayed a normal shape and density when cells were incubated with the calpain inhibitor at IC50 dose, confirming the secondary effect of MDL28170 on this organelle, as previously shown in the estimation of Dym. Some parasites incubated with two times the IC50 dose presented an empty cytoplasm and nucleoplasm and an apparent rupture of the nuclear envelope. Besides these ultrastructural changes, the detection of an altered chromatin condensation pattern in L. amazonensis promastigotes during the MDL28170 treatment is suggestive of an apoptosis-like cell death process, as described elsewhere in trypanosomatids treated with other drugs. Based on the data suggesting nuclear condensation in promastigotes, we sought to further investigate the possibility of apoptotic-like cell death mediated by the calpain inhibitor. In order to do so, promastigotes treated with MDL28170 for 72 h were double-stained with Annexin-V-FITC and PI. The percentage of promastigotes that were positive only for Annexin-V was 4.98% after treatment with the IC50 dose of MDL28170 and 22.11% when cells were treated with two times the IC50, which suggests that these cells were in the early stages of apoptosis-like death. The number of cells that were both AnnexinV- and PI-positive was 4.61% and 31.62%, respectively, which is related to late events of apoptosis-like and/or necrosis. No significant difference in the number of apoptotic-like and necrotic cells was observed after 96-h incubation. Nontreated cells were Annexin-V- and PI-negative, and the same result was seen in cells treated only with DMSO, the vehicle of drug, which confirms the viability of cells in these conditions. Alternatively, promastigotes treated with miltefosine were used as a positive control in this experiment. After 24 h of incubation with miltefosine at 40 mM, 1.02% of cells were apoptotic-like and 81.8% of cells were already in late apoptotic-like phase or necrosis. The observation of most of cells being Annexin-V-positive and PI-positive on miltefosine treatment beyond 24 h was already demonstrated to be associated to cells at a very advanced stage of apoptosis-like death and resemble necrotic cells, which are difficult to discriminate. A critical point in the analysis of the results presented in Figure 4 is concerned to the presence or lack of PS in Leishmania spp.