ThT and Bis-ANS Fluorescence Assay

ThT fluorescence assay was performed to detect the fibril formation of Aβ and mutants, as an increase in fluorescence emission at 485 nm occurs upon the amyloid-specific dye ThT incorporation into β-sheet amyloid structures (LeVine, 1999). The hydrophobic probe Bis-ANS, which can bind to hydrophobic patches formed due to aggregation and induce an increase in fluorescence emission at 496 nm (Rosen and Weber, 1969), was also used to determine the accumulation of Aβ and mutants. All the fluorescence assay was performed on a Synergy H1 multi-mode microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) with a Nunc 96-well flat-bottom, black polystyrene microplate (Thermo Fisher Scientific). For the measurement, Samples (50 μM) were incubated with 50 μM ThT or Bis-ANS in PB at 37°C for 48 h. The ThT fluorescence was measured every 60 min using an excitation wavelength at 440 nm. The Bis-ANS fluorescence was tested at 48 h using an excitation wavelength at 385 nm.