Spectramax paradigm multi mode reader

Inhibitory effects of the compounds on self-induced Aβ_1–42 aggregation were tested through a Thioflavin T (ThT)-(T3516, Sigma-Aldrich) binding assay. Firstly, aliquots of 2 μL of Aβ_1–42 (AS-64129–05 Anaspec Inc.) containing 2 mg/mL HFIP (1,1,1,3,3,3-hexafluoro-2-propanol, 52517( Sigma-Aldrich) were stocked in DMSO. Then, they were diluted with 0.215 M sodium phosphate buffer (pH 8.0) to the final concentration of 500 μM. Test compounds were dissolved in DMSO and then prepared at a concentration of 25 μM by the buffer. Resveratrol was used as a positive control. The Aβ_1–42 and the test sample solutions were incubated in a 96-well plate for 24 h at the room temperature. After the incubation, the tested compounds were diluted to a final volume of 150 μL with 50 mM glycine-NaOH buffer (pH 8.5) containing 5 mM ThT. Fluorescence intensity was read (excitation wavelength 450 nm, emission wavelength 485 nm) on a SpectraMax Paradigm Multimode Reader (Molecular Device, Sunnyvale, CA).
The calculation of the inhibitory rate of Aβ_1–42 self-induced aggregation was performed as the following equation: (1 − I_Fi/I_Fc) × 100%. I_Fi and I_Fc were the fluorescence intensities measured in the presence and absence of inhibitors, respectively, after subtracting the background fluorescence of the 5 mM ThT solution. Each compound was measured in triplicate.