Sbiii

Promastigotes of wild-type L. braziliensis and L. infantum clonal lines non-transfected or transfected with the constructs pIR1BSD (empty vector), pIR1BSD-NDKb or pIR1BSD-EF2 were submitted to Sb^III (Sigma-Aldrich, St. Louis, MO, USA) susceptibility tests. The susceptibility to hydrogen peroxide (H₂O₂) was also evaluated in the parasites transfected with the NDKb gene. Parasites were incubated in M199 medium at 2 × 10⁶ cells/ml into 24-well plates in the absence or presence of several concentrations of Sb^III (1.17 to 599.04 μM which corresponds to 0.00078125 to 0.4 mg/ml) or H₂O₂ (100 to 350 μM) for 48 h. The effective concentration required to decrease growth by 50% (EC₅₀) was determined using a model Z1 Coulter Counter (Beckman Coulter, Fullerton, CA, USA). EC₅₀ values were determined from at least three independent measurements performed in triplicate, using the linear interpolation method [38 (link)].

L. amazonensis (R0, R10, R40, and R60) promastigotes at the 4th day of cultivation were submitted to Sb^III (Sigma-Aldrich, St. Louis, MO, USA) susceptibility tests. Suspensions of parasites at 2 × 10⁶ cells/mL were incubated in 24-well plates with M199 medium at 26°C for 48 h. The parasites were incubated either in the absence of Sb^III or exposed to several different concentrations of that compound (18.7–374.3 μM). The effective concentrations required to kill 50% of the parasites (EC50) were determined using a model Z1 Coulter Counter (Beckman Coulter, Fullerton, CA, USA). EC50 values were determined from at least two independent measurements performed in triplicate, applying the method of linear interpolation (Huber and Koella, 1993 (link)). Statistical analyses were performed using GraphPad Prism Software v5.0, San Diego, CA.