Synergy h4 hybrid multi mode

The assay was performed as described previously (Nilsson, 2004 (link)). Briefly, 7 mg/mL Congo Red stock solution was prepared in 30 mM Tris buffer, pH 7.5, and filtered through 0.22 µm syringe filter and stored at room temperature for up to a week. Alpha-synuclein samples were taken at different times of aggregation. 50 µL of alpha-synuclein sample was mixed with 50 µL of 70 µg/mL Congo Red (freshly prepared from stock solution) and incubated for 30 min at room temperature in 96 well non-binding half-area plate (Corning NBSTM). Spectra were recorded using Synergy H4 Hybrid Multi-Mode (Biotek) microplate reader. In case of Abeta, 35 µg/mL of Congo Red were added to the 15 µM peptide samples at the beginning of the reaction. 100 µL aliquots were transferred into wells of a 96 well non-binding half-area plate (Corning NBS; Corning Inc., Corning, New York, USA). The plate was incubated at 37 °C temperature, and spectra were recorded at different time points using Synergy H4 Hybrid Multi-Mode (Biotek) microplate reader. To get differential spectra, the corresponding spectrum at zero time point was mathematically subtracted from the spectra at later time points.

Insulin aggregation kinetics were monitored at 60 °C without agitation by measuring ThT fluorescence emission intensity (excitation wavelength—440 nm, emission—480 nm) through the bottom of a 96 well non-binding surface plate using Synergy H4 Hybrid Multi-Mode (Biotek) plate reader (readouts were taken every 10 min to minimize plate agitation). For every condition, four independent measurements were performed. Aggregation half-times (t₅₀) were calculated as the time needed to reach 50% of signal intensity. The full concentration range of aggregated insulin samples were centrifuged at 10 000 g for 30 min and the residual unaggregated insulin in the supernatant was determined to be less than 1% of initial protein concentration.

Freshly purified (within 30 min after gelfiltration) monomeric Abeta42 solution was supplemented with 50 µM ThT and diluted using buffer E containing 50 µM ThT to reach different concentrations (1–6 µM). For the inhibition experiments 216, 36, 6, 1, and 0.167 µM solutions of flavones in buffer D, containing 2% DMSO and 50 µM ThT were prepared. Monomeric 6 µM Abeta42 solution was mixed with these flavone derivative solutions (or with buffer E containing 2% DMSO as a control) in a 1:1 ratio. Each sample was divided into three 100 µL aliquots into wells of a 96 well non-binding half-area plate (Corning NBS™). Kinetics of aggregation was followed at 37 °C temperature and constant shaking (960 rpm) using Synergy H4 Hybrid Multi-Mode (Biotek, Winooski, Vermont, USA) microplate reader. The intensity of ThT fluorescence was measured through the bottom of the plate every 3 min using 440 nm excitation and 482 nm emission.