ThT Fluorescence Assay

Thioflavin T (ThT) fluorescence assays were performed to monitor the real-time aggregation of Aβ₄₂ and Aβ₄₀ in the absence or presence of designed peptides. ThT assays were conducted in 96-well plates (black with flat optical bottom) in a Varioskan fluorescence plate reader (Thermo Scientific, 444 nm excitation, 484 nm emission). Each experiment was run in triplicates. The reaction solution contained 30 μM pre-disaggregated Aβ₄₂ or Aβ₄₀, 10 μM ThT, and designed peptides at indicated concentrations in PBS. The ThT assay was conducted at 37°C, without shaking for the Aβ₄₂ aggregation assay, and with shaking (300 rpm) for Aβ₄₀ aggregation assay. The fluorescence readings were collected every 2 min.