ThT Fluorescence Assay

ThT is a fluorescence dye that binds to the β-sheet containing amyloid structures; this leads to an increase of its fluorescence and thus enables the kinetics of amyloid formation to be followed and quantified. The ThT assay was performed as described previously.³⁶ 100 μM S100A9 was transferred into Corning 96 black well plates with transparent bottoms, and then, 20 μM ThT was added to every well. Sample volumes were kept at 200 μL per well. The 96 well plates were covered, transferred immediately into a Tecan F200 PRO plate reader, and subjected to incubation at 42 °C during 50 h by applying 432 rpm orbital shaking every 10 min. Fluorescence of ThT dye was recorded every 10 min. A filter at 430 nm was used for excitation, and a filter at 495 nm was used for emission; both filters were characterized by 20 nm band widths. All protein solutions were incubated in triplicates.