4.2. TTR Aggregation Assay

For the acid-induced aggregation assay, we followed the protocol described previously [4,64]. Briefly, TTR samples prepared in PBS buffer pH 7.4 (10 mM phosphate, 140 mM NaCl, 2.7 mM KCl) were first mixed with either DMSO, or equimolar, and five-fold test compounds (GC-1, IS25, or TG68) dissolved in DMSO. These TTR-compound mixtures were then mixed 1:1 with acetate buffer (200 mM sodium acetate pH 4.2, 100 mM KCl, 1 mM EDTA) to make the final solution of pH 4.4 [64]. The final monomer concentration of TTR was 64 μM. The mixtures were incubated at 37 °C for four days without agitation.

The aggregation level was quantified either by ThT fluorescence or turbidity, both of which were measured with a Tecan Spark™ 10M microplate reader (Männedorf, Switzerland). For ThT fluorescence measurement, the aggregation mixtures were first diluted to the final concentration of 4 μM TTR with a buffer (200 mM Tris pH 8.0, and 150 mM NaCl). Next, 2 μL 2 mM ThT stock solution in 200 mM Tris pH 8.0, 150 mM NaCl was added to 400 μL of the diluted solution. The final samples were vortexed and transferred to a 96-well black-wall microplate in triplicate for the ThT fluorescence measurement (excitation and emission wavelengths were set to 440 nm and 482 nm, respectively). The average fluorescence values were reported as mean ± SD from triplicate measurements. For the turbidity measurement, the aggregation mixtures were vortexed and pipetted into a 96-well UV-compatible transparent microplate in triplicate. The turbidity of the samples was measured by recording the optical density at 330 nm, and the mean ± SD was reported. We confirmed the consistency of the ThT fluorescence and turbidity results by repeating the measurements several times.