Thioflavin T is a fluorescent dye that binds to beta sheet-rich structures, such as those in tau fibrils. Upon binding, the emission spectrum of the dye experiences a red-shift and increased fluorescence intensity. The following protocol was used to generate the Thioflavin T assays using tau pre-formed fibrils and monomers.

1. A 1mM stock solution of Thioflavin T was prepared in dH2O (prepared fresh and filtered through a 0.2 µm syringe filter).
2. The thioflavin T was diluted in PBS pH 7.4 so that the final Thioflavin T concentration in each well was 25 µM (volume per well = 100 µL).
3. 10 μM Heparin was added to each well.
4. Tau aliquots were thawed at 37°C just before use.
5. Either fibril or monomer (or both) was added to the appropriate wells. The well contents were pipetted up and down to mix.
6. The plate was sealed and placed in a shaking incubator (800 rpm) at 37°C.
7. Fluorescence was measured on a Molecular Devices Gemini XPS Microplate Reader using Softmax Pro software version 6.5.1.
8. The plate was re-sealed and placed into the shaking incubator at 37°C.
9. Readings were taking at regular intervals from 1 hour to 72 hours.

 

Plate: Greiner-Bio 96 Well Non-Binding Cell Culture Microplate, Black (Greiner-Bio Catalog #655900).

 

XPS Microplate Reader Settings:

Temperature: 37°C

Read Type: Well Scan

Wavelength: Excitation at 450 nm and Emission at 485 nm

PMT Gain: Automatic

Flashes per read: 6

Shake: 20 seconds before read