Schneider medium

L. braziliensis promastigotes strain MHOM/BR/01/BA788 were grown in Schneider medium (Sigma) supplemented with 100U/ml of penicillin, 100ug/ml of streptomycin, 10% heat-inactivated fetal calf serum (all from Life Technologies). Stationary-phase promastigotes (10⁵ parasites in 10μl of saline) were inoculated into the right ear dermis of age-matched BALB/c mice using a 27.5-gauge needle. Lesion size was monitored weekly, for 5 weeks, using a digital calliper (Thomas Scientific).

L. braziliensis promastigotes (strain MHOM/BR/01/BA788) were grown in Schneider medium (Sigma), supplemented with 100 U/ml of penicillin, 100 ug/ml of streptomycin and 10% heat-inactivated fetal calf serum (all from Invitrogen).

Leishmania amazonensis (WHOM/BR/Josefa) and Leishmania major (MHOM/IL/80/Friedlin) were grown in Schneider medium (Sigma-Aldrich) supplemented with 10% fetal calf serum and 1% penicillin–streptomycin at 26 °C The stationary phase promastigotes were washed three times with PBS (1045 g, 10 min, 4 °C) and, resuspended in medium RPMI 1640. L. major promastigotes were washed with PBS and incubated for 15 min with PNA lectin (Sigma) to separate the metacyclic form. 4% formaldehyde was used for parasite fixation, followed by two washes with PBS.

The amastigote cultures of L. amazonesis MHOM/BR/pH8 strain were kept under cryopreservation until transferred to NNN medium (Novy–MacNeal–Nicolle) and cultured at 26 °C until the parasites reached the log growth phase. Then, the suspension was transferred to Schneider culture medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% inactivated fetal bovine serum and 0.2% gentamicin sulfate, at 26 °C, so that the parasites returned to the log phase of growth. After that stage, the promastigote forms were incubated at 37 °C, producing a growth curve and the formation of the axenic amastigote strains [79 (link)].
The promastigote forms of L. amazonensis MHOM/BR/pH8 strain were preserved in Schneider medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 20% fetal bovine serum (FBS) and 1% streptomycin/penicillin, at 26 °C, in a B.O.D incubator—J. Prolab, São José dos Pinhais, Brasil, model JP. 100 (LBCM/CPAM). The promastigote forms were used in the exponential growth phase in all stages of the experiment [80 (link)].

L. amazonensis (MHOM/BR88/BA-125 Leila strain) and L. braziliensis (MHOM/BR88/BA-3456) promastigotes (1 × 10⁶ per well) were cultured in a 96-well plate in Schneider medium (Sigma-Aldrich^®, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO) and 50 μg mL⁻¹ gentamicin (Life, Carlsbad, CA, USA) and subjected to treatment with different concentrations (100 μM; six dilutions 1:2) of Q5. The parasites were incubated for 72 h at 26 °C. Then, 20 µL/well of AlamarBlue (Invitrogen, Carlsbad, CA, USA) was added over 2 h. The reading was carried out in a spectrophotometer using the wavelengths of 570 and 600 nm. The calculation of axenic culture inhibition was determined based on the untreated control [49 (link)].