Spectramax m2 m2e

Cells were seeded at appropriate densities (adherent cell lines: 5,000 cells per well; mixed-adherent cell lines: 10,000 cells per well) and allowed to incubate overnight at 37 °C. Cells were treated with serial dilutions of the drug and incubated for 72 h. Cell counting kit-8 (WST-8/CCK8) reagent from GLPBIO was added to each well and allowed to incubate for 2 h. The absorbance was measured at 450 nm on a SpectraMax M2/M2E microplate reader (Molecular Devices). Final cell viability was determined relative to the average absorbance of solvent control-treated cells.

For analysis of lipid bodies accumulation, 1.0 × 10⁷ cells were harvested, washed in PBS (pH 7.2), and incubated with 10 µg/ml Nile Red (Sigma, Brazil) for 20 min. After this step, cells were washed twice in PBS, resuspended in 200 µl of PBS, and then added to a black 96-well plate. Readings were taken in a microplate reader and spectrofluorometer SpectraMax M2/M2^e (Molecular Devices, United States), using the wavelengths 485 and 538 nm for excitation and emission, respectively.

Cytotoxicity of the isolated or extracted compounds was assessed through a colorimetric assay. Approximately 1 × 10⁵ cells/mL were seeded in 96-well plates and incubated with the compounds for 24 h. MAPK inhibitors were administered 2 h before the compounds were assessed. Thereafter, 100-μg/mL MTT was added to each well. After 2.5-h incubation at 37 °C, the supernatants were aspirated, and cells were treated with DMSO to dissolve the formazan crystals. Absorbance of the colored solution was determined at 540 nm using a SpectraMax M2/M2e (Molecular Devices, San Jose, CA, USA).

We seeded 2 × 10⁴ RAW 264.7 cells/mL in a 96-well plate. After 24 h, the cells were incubated with various concentration of IPA (2.5, 5, 10, 15, and 20 μg/mL) for 5 days. MG-63 cells were seeded in a 96-well plate at a concentration of 1.5 × 10⁴ cells/well, and treated with various concentration of IPA (3.125, 6.25, 12.5, and 25 μg/mL) for 3 days. MTT assay were performed according to the method of Cho et al. [35 (link)]. Absorbance of the formazan crystals was measured at 540 nm using a SpectraMax M2/M2e spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Mitochondrial transmembrane electric potentials (ΔΨm) of Leishmania amazonensis control and treated promastigotes were evaluated using the JC-1 fluorochrome (Molecular Probes, United States)^{6 (link)–8 (link)}. Initially, cells were washed in PBS, pH 7.2, and resuspended in a mitochondrial reaction medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES/K+, pH 7.2, 2 mM propidium iodide (Pi), 1 mM MgCl₂ and 500 µM EGTA. Then, 1.0 × 10⁷ parasites were incubated with 10 µg/mL JC-1 for 40 min at 25 °C, with readings made every minute using a microplate reader and spectrofluorometer SpectraMax M2/M2e (Molecular Devices). After 36 min, 2 µM FCCP was added to abolish the ΔΨm sustained at the inner mitochondrial membrane by the respiratory chain. Relative ΔΨm was obtained by calculating the ratio between the reading at 590 nm and the reading at 530 nm (590:530 ratio). Results obtained from each triplicate were analyzed and subjected to statistical analysis using Prism 4 (GraphPad software). Statistical significance was assessed using the one-way analysis of variance (ANOVA) test followed by Bonferroni’s multiple comparison test in the GraphPad Prism 4 Software. Results were considered statistically significant when p < 0.05.

The copper chelation test was performed as described previously [51 (link)]. Briefly, 96-well microplates with a reaction mixture containing different concentrations of samples (0.1–2 mg/mL), pyrocatechol violet (4 mM) and copper II sulfate pentahydrate (50 mg/mL) were homogenized with the aid of a micropipette, and the absorbance of the solution was measured at 632 nm using a microplate reader (SpectraMax^® M2/M2e, Molecular Devices, São José, CA, USA).