Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader

Mix reagents to a final concentration of 10micromolar (µM) Tau (TauRD), 2.5micromolar (µM) Heparin, 2millimolar (mM) MgCl2, 10micromolar (µM) ThT in 1x PBS 7.2. Prepare a mix for 4.5 reactions per condition (per condition, four technical replicates in each plate). Final volume is 80µL per well.

Dispense 80µL of the mix into a well of a 96 well half-area plate of black polystyrene with a clear bottom (Corning 3881).

If possible, the outer wells should not be used and instead filled with water.

Seal the plate with parafilm to avoid evaporation.

Set the following parameters in a SPARK multimode microplate reader (TECAN) and start the reaction:

• Fluorescence measurement: ThT signal, excitation 440 nm, emission 480 nm, (use gain regulation) measured every 2 minutes.
• Temperature 37°C
• Constant shaking (50 seconds linear shaking: amplitude 4.5 mm, frequency 420 rpm - 50 seconds orbital shaking: amplitude 1.5 mm, frequency 360 rpm).
  Note

  SPARK multimode microplate reader (TECAN) is highly recommended due to its high sensitivity and the gain regulation mode that increases the fluorescence detection window. When using other plate readers, the sample ThT signal easily gets saturated even when reducing initial gain to the minimum gain capacity. Cysteine-free TauRD (Tau residues 244-371, C291A/P301L/C322A/V337M), rapidly aggregates under these conditions, reaching the ThT plateau after 2 h aggregation.