96 well black optical bottom plate

The RT-QuIC was performed as described previously [21 (link), 81 (link)] with minor modifications. Recombinant PrP was seeded with clarified 10% w/v brain homogenates lysed in PBS 0.1% SDS and diluted 10–8 in PBS in a 96-well black optical bottom plate (Fisher-Scientific). Each sample was run in duplicate. Prepared plates were sealed (VWR) and incubated in a FLUO Star OPTIMA plate reader (BMG Labtech) at 42°C for 80 h with intermittent shaking cycles, consisting of one minute double orbital shaking at the highest speed (600 rpm) followed by a 1-min break. Beta-sheet formation kinetics was determined by measuring the Thioflavin-T (ThT) fluorescence signal (450 nm excitation and 480 nm emission) every 30 min in relative fluorescence units (rfu). In vitro proteolytic assays with active human Calpain 1 (Millipore) were performed on 1% (w/v) brain lysates for 30 min at 37°C in buffers recommended by commercial suppliers.

The RT-QuIC was performed as reported before6 (link)8 (link). 85 μL of reaction buffer (consisting of 5xPBS (pH 6.9), 170 mM sodium chloride (SAFC Biosciences), 1 mM EDTA (Sigma-Aldrich), 10 μM Th-T (Sigma-Aldrich), and 0.1 mg/mL recPrP^C (Thermo Fisher Scientific/Prionics) was seeded with 15 μL CSF or 15 μL of brain homogenate at a dilution of 10⁻³ to a final volume of 100 μL in a 96-well black optical bottom plate (Fisher-Scientific). Each patient sample (brain and CSF) was analysed in triplicate. Prepared plates were sealed with sealing tape (Nunc/Sigma-Aldrich) and incubated in a FLUO Star OPTIMA plate reader (BMG Labtech) at 42 °C for 80 hours with intermittent quaking cycles, consisting of one-minute double-orbital quaking at 600 rpm followed by a one-minute incubation break. Beta-sheet formation kinetic was determined by measuring the Th-T fluorescence signal (450 nm excitation and 480 nm emission) every 30 minutes in relative fluorescence units (rfu).

RT-QuIC was performed as previously described [101 (link)] with minor modifications. Briefly, recombinant PrP (10 μg) was seeded with 15 μl of Ago-2-Immunoprecipitates in 85 μl of reaction buffer. Reaction was set in a final volume of 100 μl and placed in a 96-well black optical bottom plate (Fisher Scientific). Each sample was run in duplicate. Prepared plates were sealed and incubated in a FLUO Star OPTIMA plate reader (BMG Labtech Ortenberg, GE) at 42°C for 80 h, with intermittent shaking cycles consisting of 1 min double orbital shaking at the highest speed (600 rpm) followed by a 1 min break.