*2.2. Parasite Culture and Growth Curve*

The *L. braziliensis* strains IOC/L2853 (MHOM/BR/2004/LTCP 393) and IOC/L2856 (MHOM/BR/2003/LTCP 15171) used in this study were provided by the Collection of *Leishmania* of the Instituto Oswaldo Cruz (CLIOC, http://clioc.fiocruz.br/ (accessed on 2 March 2020)). The IOC/L2853 strain is resistant to NO and was isolated from a MCL patient, who was refractory to treatment. On the other hand, the IOC/L2856 strain is susceptible to NO and was isolated from LCL patient responsive to the treatment [17,20]. Both strains were isolated from the same geographical region and belong to the same zymodeme. Through the text, tables, and figures, those strains will be mentioned as 2853 and 2856.

Promastigotes were cultivated at 25 ◦C in Schneider's medium (Vitrocell, Campinas, Brazil) supplemented with 20% fetal bovine serum (FBS; Vitrocell, heat-inactivated at 56 ◦C for 50 min) and 2% urine. To analyze the growth curve of these strains, promastigotes (1 × <sup>10</sup><sup>5</sup> parasites) were incubated in the culture medium described above, and parasite density was determined every 24 h during 16 days by counting in hemocytometer under light microscopy. Parasites of three-days-old culture (log phase promastigotes) were used for all experiments. The in vitro passages of parasites were controlled, and parasites' infectious capacity was maintained through inoculation in golden hamsters.
