*2.3. Inhibitory Activity of NO Donor on Promastigotes of L. braziliensis*

To evaluate the cytotoxicity of NaNO2 (NO donor) on NO-resistant (2853) or NOsusceptible (2856) *L. braziliensis* strains, promastigotes were resuspended in Hanks balanced solution (HBBs, pH 5.0; Sigma-Aldrich, St. Louis, MO, USA), and 100 <sup>μ</sup>L (2 × 107 protozoa/mL) was added to the same volume of freshly NaNO2 previously prepared at twice the desired final concentrations (32 to 0.06 mM). Microplates were incubated at 25 ◦C for 4 h. Together with the NO donor, 20 μL of PrestoBlue (Invitrogen, Carlsbad, CA, USA) was added to the final concentration of 10%. The measurements were performed at 560 and 590 nm, as recommended by the manufacturer at SpectraMax M3 fluorimeter (Molecular Devices, San Jose, CA, USA), and the result was expressed as IC50/4 h, which corresponds to concentration that led to 50% lysis of the parasites within 4 h.
