2.2. Parasites

Leishmania amazonensis (MHOM/BR/75/Josefa) was purified from the lesions of infected BALB/c mice and maintained in M199 medium (Sigma-Aldrich, San Luiz, MI, USA) supplemented with 0.02% hemin, 10% heat-inactivated fetal bovine serum (FBS) (Cultilab, Campinas, São Paulo, Brazil), 100 U/L penicillin, and 100 μg/L streptomycin at 26 °C prior to the in vivo experimental infections. For in vivo infection, promastigotes in the stationary phase of growth were used, promastigotes were washed three times in phosphate buffered saline (PBS) at 800× g for 10 min, and the pellet was resuspended in PBS. To ensure infectivity, the parasites were only used in experiments up to the fifth passage in in vitro culture.