Spectramax m5 spectrofluorometer

T. cruzi epimastigotes of the INC-5 strain were used to obtain a protein extract quantified by the Bradford method. Evaluation of the activity was carried out using the synthetic fluorogenic peptide Z-Phe-Arg-MCA as a substrate at 5 µM, with 1 μg of the enzyme extract and the corresponding compounds (Z2, Z3, Z5, and Z6) at different concentrations (6.25–200 µM) using a reaction buffer (50 mM Na₂HPO₄, 100 mM NaCl, 5 mM EDTA, 2.5 mM dithiothreitol; pH 6.5) at room temperature. As a negative control of enzyme inhibition, the substrate without compounds was evaluated with 2% DMSO (concentration corresponding to the highest dilution of the compounds). Hydrolysis of the substrate was monitored in a continuous assay for 1 h in the Spectramax M5 spectrofluorometer (Molecular Devices) at 380 nm excitation and 440 nm emission. The mean of fluorescence changes was normalized taking the control (without inhibitor) as 100%. The IC₅₀ values were determined from semilog concentration-response plots using the nonlinear curve-fitting method [41 (link)].
The relationship of the biological evaluation with the binding mode of the selected molecules was inferred based on in vitro results.

Dichlorofluorescindiacetate (DCFH-DA) was used to qualitatively detect the formation of ROS. Non-fluorescent DCFH-DA is oxidized to fluorescent dichlorofluorescein (DCF) by ROS. Cells were plated at 1 × 10⁵ in a confocal dish and incubated for 24 h. Cells were treated with vehicle (0.9% saline) and PolyHb and further incubated with DCFH-DA (10 μM) for 15 min before imaging. Glass dishes were observed by confocal laser scanning microscopy (CLSM). Quantitative analysis of intracellular ROS was conducted after cells were digested and collected by centrifugation (1000 rpm, 5 min), and quickly the fluorescence intensity was measured using a Spectra Max M5 spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) at an excitation of 485 nm and emission of 535 nm.