MTT cell proliferation assay

The MTT assay, adapted for Leishmania spp. 22, was used to assess promastigote viability in different experiments. Briefly, promastigotes were pelleted by centrifugation and washed twice in PBS (137 mm NaCl, 2.7 mm KCl, 10 mm Na₂HPO₄ and 1.8 mm KH₂PO₄, pH 7.4). Finally, parasites were resuspended in PBS, to which 5.0 mg·mL⁻¹ of MTT was added. After incubation for 60 min at 25 °C, 0.1 mL of a 10% of SDS lysis solution was added to stop the reaction 22. Absorbance was monitored using a fluorometer POLARstar^® Omega (BMG Labtech, Waltham, MA, USA), at 595 nm and 690 nm as reference. The MTT methodology was used to perform three kinds of experiments as described below.

We studied the antileishmanial activity of two HPPD inhibitors. Promastigotes in late logarithmic phase were treated with various concentrations (2.5 μm to 0.078 μm) of usnic acid (UA) or nitisinone (NTBC) (250 μm to 3.9 μm), and parasite viability was evaluated by MTT assay. Control cultures were treated with DMSO and/or acetone as solvent controls, and their effects on parasite viability were assessed daily by MTT measurements as well. The inhibitor concentration where the growth is reduced by half (IC₅₀) was calculated after 72 h of treatment.

In the second step, we studied whether tocopherol could recover HPPD inhibitor effects on parasite viability. Using a concentration of usnic acid or nitisinone where the growth is reduced near by half, promastigotes in late logarithmic phase were treated for 72 h with 0.5 μm usnic acid or for 48 h with 31.25 μm NTBC with or without exogenously added 25 μm α‐tocopherol in the complete medium and the parasite viability was evaluated by MTT methodology.

In the third step, we studied staurosporine and tocopherol interactions. Promastigotes in late logarithmic phase were treated with staurosporine 75 nm in TC‐100 medium with or without various concentrations of α‐tocopherol (30, 60 or 90 μm). We previously observed that the described staurosporine concentrations and periods of treatment produce viability decreases. After 1‐h incubation at 25 °C, the cells were washed twice and the culture was recovered in complete TC‐100 medium. Then, the treatments effects on parasite viability were assessed by MTT measurements.