In L. amazonensis, an 80-kDa calpain-like protein was identified on the cell surface of the parasite using an anti-calpain antibody developed against D. melanogaster calpain, and no crossreactivity was found with mammalian calpains. With these results in mind, we aimed to investigate in the present work the mechanism of cellular death promoted by this compound in L. amazonensis promastigotes. Through the combined use of different techniques, we found that MDL28170 induces the expression of apoptotic markers in these cells. The effects of MDL28170 on promastigote forms of L. amazonensis were order 220904-83-6 assessed by a method similar to that described previously. Promastigotes were counted using a Neubauer chamber and resuspended in fresh medium to a final concentration of viable promastigotesl. The calpain 29700-22-9 inhibitor was added to the culture at final concentrations ranging. Dilutions of DMSO corresponding to those used to prepare the drug solutions were assessed in parallel. After of incubation the number of late-log, viable motile promastigotes was quantified in a Neubauer chamber. This incubation period was chosen because a significant reduction in the growth rate was observed for late-log phase promastigotes in comparison to mid-log phase cells. The inhibitory concentration, the minimum drug concentration that caused a reduction in survival/viability was determined by linear regression analysis by plotting the number of viable promastigotes versus log drug concentration using Origin computer software. Resazurin is a redox potential indicator that is converted to fluorescent and colorimetric resorufin dye by the metabolically active cells. Non-viable cells rapidly lose their metabolic capacity to reduce resazurin in the mitochondrion and, thus, do not produce fluorescent signals anymore. Assays were performed in sterile 96-well plates using late log-phase promastigotes in the absence or in the presence of the IC50 or two times the IC50 doses of MDL28170. After incubation of resazurin were added, and plates were incubated for a further at the same temperature. After incubation, cells were analyzed at a microplate reader using a pair as emission and excitation wavelengths, respectively. The viability was evaluated based on a comparison with untreated, control cells. Parasites were also treated with sodium azide for 30 min i
