Optical bottom black 96 well plates

All compounds were assayed in vitro against L. infantum BCN150 iRFP promastigotes (iRFP-L. infantum), a genetically modified strain that constitutively produces the infrared fluorescent protein (iRFP) for near infrared detection (Calvo-Álvarez et al., 2015b ). Promastigotes were cultured in M199 medium (Gibco), supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 6.9, 7.6 mM hemin, 10 mM glutamine, 0.1 mM adenosine, 0.01 mM folic acid, 1xRPMI 1640 vitamin mix (Sigma), 10% (v/v) heat inactivated foetal bovine serum (FBS) (Gibco), 50 U/mL penicillin and 50 μg/mL streptomycin. Cultures of iRFP-L. infantum promastigotes with a density of 1 × 10⁶ cells/mL, were dispensed into 96-well optical bottom black plates (Thermo Scientific), 180 μL per well. Each compound was tested adding 20 μL of different stock solutions to the inoculated wells. Stock solutions were prepared in dimethylsulfoxide (DMSO) and serially diluted in M199 media (0.01–200 μM final concentrations). The viability of promastigotes to calculate the 50% effective concentration (EC₅₀) values was assessed measuring their fluorescence at 713 nm in an Odyssey (Li-Cor) infrared imaging system after 72 h exposure at 26 °C. All compounds and controls were assayed by triplicate. Plots were fitted by nonlinear analysis using the Sigma Plot 10.1 statistical package.