H2dffda

The oxidative fluorescent dye H₂DFFDA (Invitrogen, Eugene, OR, USA) was used to evaluate the ROS production induced with compound 8. Leishmania mexicana promastigotes were treated with compound 8 (IC₅₀ value) for 24 h and 48 h at 26 °C and centrifuged at 327× g for 10 min. Then, 1 × 10⁷ parasites/mL were incubated with 200 µL of H₂DFFDA (10 µM) for 60 min in the dark at room temperature. Hydrogen peroxide (100 µM) and non-treated parasites were used as positive and negative controls, respectively. The H₂DFFDA-fluorescence intensity was measured at 485 nm excitation and 538 nm emission wavelength with a cytofluorimeter (Fluoroskan Ascent FL, Thermo Labsystems, Waltham, MA, USA).

BMDM and neutrophils were incubated with HKCAs (at a multiplicity of infection of 1:20) for 0, 2, 4 or 6 hours, to measure the total intracellular ROS levels, cells were treated with the fluorogenic probe H2DFFDA (Life Technologies) at 5 μM for 30 min at 37°C. The medium was then removed, and the cells were returned to prewarmed fresh growth medium. The emitted fluorescence was detected by a fluorescent microplate reader using 490/520 nm excitation/emission filters (Molecular Devices, Sunnyvale, CA). The ROS levels are reported as fluorescence intensity.