CL Brener clone T. cruzi epimastigotes were maintained axenically at 28 °C in LIT medium supplemented with 10% fetal calf serum (FCS) with weekly transfers. Four-day old cultures at the mid-log phase of growth were used in all experiments. The tissue culture trypomastigotes were obtained from the supernatants of 5- to 6-day old infected LLC-MK2 cells that were maintained in RPMI-1640 medium supplemented with 2% FCS for 5–6 days at 37 °C in a 5% humidified CO2 atmosphere, as previously described

( Adade et al., 2011). The intracellular amastigotes were obtained and cultured PI3K inhibitor as described below. The melittin peptide was purchased from Sigma Chemical Co. (St. Louis, MO, USA). A stock solution (250 μg/ml) was prepared in phosphate-buffered saline (PBS, pH 7.2) and stored at −20 °C until further use.

We initially relied on the data obtained from the trypanocidal activity of A. mellifera crude venom ( Adade et al., 2012) to define the ranges of melittin concentrations to be tested. The epimastigotes were resuspended in LIT medium at a concentration of 1 × 106 cells/ml and incubated with 1.34–5.36 μg/ml of melittin in a 24-well plate (Nunc Inc., Naperville, IL, USA). They were then incubated for 96 h at 28 °C. The number of parasites was determined daily PCI-32765 datasheet by counting formalin-fixed parasites in a hemocytometer. The parameter used to estimate the inhibition of proliferation was the IC50, which represents the drug concentration that inhibited 50% of cell growth. The parasites grown medroxyprogesterone in drug-free LIT medium were used as controls. The growth experiments were carried out in triplicate, and the standard deviation of the cell densities at each time point was presented with error bars. The cell viability was verified by the detection of propidium iodide staining by flow cytometry (described below). The tissue culture trypomastigotes (1 × 106 cells/ml) were resuspended

in RPMI media (Sigma) containing 10% FCS and incubated with 0.1–0.4 μg/ml of melittin in a 96-well plate (Nunc Inc.). This was followed by incubation at 37 °C. The LD50 parameter (50% trypomastigote lysis) was evaluated by counting the formalin-fixed parasites in a hemocytometer after 24 h. The experiments were performed in triplicate. The uninfected LLC-MK2 cells were seeded in 24-well plates (Nunc Inc.) containing glass coverslips. They were maintained in RPMI media supplemented with 10% FCS and were treated or not with 1 and 5 μg/ml of melittin at 37 °C for 72 h. The cytotoxic effects were examined daily using a trypan blue exclusion test. Briefly, at the end of the incubation period, the glass coverslips were washed with sterile PBS (pH 7.2) and stained with a 1:1 dilution of trypan blue solution:RPMI for 5 min.