Thioflavin T fluorescence to analyse amyloid formation kinetics: Measurement frequency as a factor explaining irreproducibility

The most frequent method to monitor amyloid formation relies on the fluorescence of thioflavin T (ThT). The present study reports a novel factor of irreproducibility in ThT kinetic assays performed in microplate. Discrepancies among kinetics of amyloid assembly, performed under quiescent conditions, were associated with the frequency of fluorescence measurement. Evaluating self-assembly of the islet amyloid polypeptide at short intervals hastened its fibrillization. This observation was confirmed by transmission electron microscopy, circular dichroism spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence. This effect, attributed to agitation during microplate displacements between fluorescence measurements, reinforces the importance of a better standardization in amyloid formation assays.