Aβ(M1-42) fibrils were generated by incubating 17 μM monomeric Aβ(M1-42) in 20 mM sodium phosphate buffer at pH 8.0 in a 96-well plate in a FLUOstar OPTIMA plate reader (BMG Labtech) at 37°C with double orbital rotation (400 rpm). Aggregation was monitored by following the increase in the fluorescence emission of a similar sample implemented with 20 μM ThT dye initiated upon its binding to amyloid fibrils. After completion of the aggregation reaction, the fibrils were collected, supplemented with Alexa Fluor 488–labeled clusterin in the concentration range between 0 and 4.5 μM, and incubated for at least 2 days at 21°C to ensure that equilibrium was obtained. Diffusion sizing measurements were then performed at 21°C, as described below. In a second set of experiments, three more points were evaluated with the addition of BSA (at the same concentration as used in the clusterin experiments, i.e., 0.8, 2, and 4.5 μM) added to a mixture of clusterin and fibrils, as described above, to examine the specificity of the binding with 17.5 μM Aβ(M1-42) fibrils. The samples were again analyzed by microfluidic diffusion methods.

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