ThT (Sigma, product no. T3516) was dissolved in PBS buffer and was filtered through a 0.2 µm syringe filter. Then the concentration of thioflavin T was determined using an extinction coefficient of 36 mM−1 cm−1 at 412 nm [27 ,28 (link)].
For measurement of ThT fluorescence without proteins, various concentrations of ThT were prepared in PBS using serial dilution. Forty microlitres of ThT sample at each concentration was transferred to a black 384-well Nonbinding Surface microplate with clear bottom (Corning product no. 3655). The ThT fluorescence was measured at room temperature (approx. 24°C) using a Victor 3 V plate reader (Perkin Elmer) through the bottom of the plate with excitation filter of 450 nm and emission filter of 490 nm.
For measurement of ThT fluorescence in the presence of amyloid fibrils, various concentrations of sonicated Aβ40, Aβ42 and Ure2 fibrils were also prepared using serial dilution. Then 20 µl of ThT was mixed with 20 µl of sonicated fibrils to achieve desired concentrations of ThT and amyloid. Fluorescence measurements were performed as described above.
For measurement of ThT fluorescence without proteins, various concentrations of ThT were prepared in PBS using serial dilution. Forty microlitres of ThT sample at each concentration was transferred to a black 384-well Nonbinding Surface microplate with clear bottom (Corning product no. 3655). The ThT fluorescence was measured at room temperature (approx. 24°C) using a Victor 3 V plate reader (Perkin Elmer) through the bottom of the plate with excitation filter of 450 nm and emission filter of 490 nm.
For measurement of ThT fluorescence in the presence of amyloid fibrils, various concentrations of sonicated Aβ40, Aβ42 and Ure2 fibrils were also prepared using serial dilution. Then 20 µl of ThT was mixed with 20 µl of sonicated fibrils to achieve desired concentrations of ThT and amyloid. Fluorescence measurements were performed as described above.
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