Thioflavin-T Fluorescence Assay

Thioflavin-T (ThT) stock solution was freshly prepared prior to each experiment by dissolving the ThT dye in autoclaved Milli-Q water at a concentration of 30 µM. The aggregation kinetic profiles of all samples described above were obtained using ThT fluorescence measurements at specific time-points during the aggregation process. At each time-point, peptide aliquots (70 µL) were mixed with ThT stock solution (70 µL) in black flat-bottom 96 well-plates (Nunclon Delta-Treated, Thermo Fisher Scientific). Immediately after mixing, ThT fluorescence intensities (λex 440 nm and λem 485 nm) were measured via an Infinite 200 Pro microplate reader (Tecan Trading AG, Switzerland). For each experiment, the ThT signal intensities represent the mean of at least triplicate measurements. The fluorescence value of the blank containing ThT only was measured and included in the calculations to produce all ThT data in Figure 1 and Figure 6A.

Kinetic profiles of the co-aggregation (IAPP-Aβ40 equimolar mixture) and self-aggregation (IAPP alone and Aβ40 alone) pathways. The self-aggregation profiles of IAPP alone and Aβ40 alone were acquired at three increasing concentrations: (A) 10 µM, (B) 20 µM and (C) 40 µM. The co-aggregation profiles of equimolar mixture of IAPP:Aβ40 were acquired at (B) 10 µM:10 µM and (C) 20 µM:20 µM. (D) ThT fluorescence intensities of the freshly prepared samples (i.e., 0-Hour) of IAPP alone, Aβ40 alone and IAPP-Aβ40 at all examined concentrations. (E) Time evolution of inter-peptide hydrogen bonds formed between IAPP and Aβ40 in their hetero-dimer system. All ThT fluorescence data (a.u.) represent mean ± SE (n = 3).

IAPP-Aβ40 co-aggregation kinetic profiles in the absence and presence of EGCG. (A) IAPP-Aβ40 (20 µM:20 µM) samples were co-incubated with increasing EGCG concentrations (10, 20, 40, and 100 µM) and at the indicated time-points, the ThT fluorescence measurements were acquired. Data represent mean ± SE (n = 3). Time evolution of inter-peptide hydrogen bonds formed between IAPP and Aβ40 in the absence (B) and presence (C) of five EGCG molecules. Time evolution of inter-molecular hydrogen bonds formed between the five EGCG molecules and each of IAPP (D) and Aβ40 (E) in the hetero-dimer system.

For the ThT-screening experiments of candidate polyphenolic inhibitors, additional control experiments were performed (experimental design was adopted from the study by (Hudson et al., 2009) to test whether the selected polyphenols interact with the ThT dye and interfere with its fluorescence spectra and/or competitively bind with its fibril binding sites. In the control experiments, the ThT fluorescence spectra (λex 440 nm, λem 470–700 nm) were acquired for pre-formed IAPP fibrils (40 µM) or Aβ40 fibrils (40 µM) in the presence of ThT (30 µM). Next, each polyphenol (40 µM) was added to the mixture of ThT (30 µM) and pre-formed IAPP fibrils (40 µM) or Aβ40 fibrils (40 µM) and the fluorescence spectra (λex 440 nm, λem 470–700 nm) were reacquired immediately to examine any changes in the ThT fluorescence spectra in very short periods of time.

For the inhibition of co-aggregation kinetic experiments, similar control experiments were acquired where EGCG (0.1–100 µM) was added to the mixture of ThT (30 µM) with preformed IAPP-Aβ40 hetero-aggregates to check if EGCG affects their ThT fluorescence spectra.