4.2.4. Thioflavin T Aggregation Assay

Thioflavin T aggregation assays were carried out in 96-well dark plates in a Tecan Infinite M200PRO Plex plate reader at 30 ◦C with cycles of 30 s of orbital shaking at 140 rpm and 10 min of rest throughout the incubation. ThT fluorescence measurements were taken every 11 min, using excitation wavelength of 450 nm and recording fluorescence emission at 480 nm.

Samples contained 0.01 mM proteins in 20 mM sodium phosphate bu ffer at pH 7.4 and 50 mM NaCl (with 0.02% NaN3 and protease inhibitors with EDTA), incubated with equimolar amount of heparin and ThT. Samples containing tau protein variants were filtered through a 100 kDa cut-o ff filter (Sartorius) to remove pre-existing large oligomers. Each measurement was performed in three replicates. The aggregation curves were analyzed by fitting each individual experimental data set with the following sigmoidal function [48]:

$$y = y\_i + \frac{y\_f}{1 + e^{-\left[\left(t - t\_{0.5}\right)/\tau\right]}}$$

where *y* is the fluorescence intensity as a function of time *t*, *y*i and *y*f are the intercept of the initial and final baselines with the *y*-axis, *t*0.5 is the time needed to reach halfway through the elongation phase and τ is the elongation time constant. The lag time is defined as *<sup>t</sup>*lag = *t*0.5 − 2τ. The values reported in thetextcorrespondtothemean ± SDoftheindividualvaluescomputedseparatelyoneachcurve.

Analysis and figures production were carried out with GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA).
