Microplate adhesive film

RT-QuIC was conducted according to the method described elsewhere with modifications⁴⁴ (link)^,45 (link). Ten µg mouse or bank vole recPrP, prion seeds (5 × 10⁻⁶ diluted prion-ill rodent or human patient brain material), and compounds for anti-prion activity tests were mixed in 100 µl of RT-QuIC buffer [0.5M NaCl, 10 µM EDTA, 10 mM phosphate buffer (pH 7.4) (Sigma-Aldrich), 10 µM ThT, 0.002% SDS]. The source of prion seeds was FVB mouse-adapted RML scrapie prions, Syrian golden hamster-adapted Hyper TME prions and sporadic CJD prions deposited in Korea CJD Diagnostic Centre (Seoul, Korea). Prion seeds were prepared from 10% (w/v) homogenate of perfused brains by serial dilution with PBS. The reaction mixture in a 96-well Black/Clear Flat Bottom Polystyrene Not Treated Microplate (Corning) sealed with a microplate adhesive film (USA Scientific) was incubated at 42 °C for 60–80 h with shaking at 700 rpm in FLUOstar Omega Fluorescence reader (BMG Labtech, Ortenberg, Germany). Fibrils formation was measured every 1 h by bottom reading of ThT fluorescence intensity using a 450 nm excitation and 480 nm emission filter.

PAFA was conducted according to the method described elsewhere³⁹ (link). Ten µg mouse recPrP was denatured to unfolded using 6 M guanidine hydrochloride (GdnHCl) (Sigma-Aldrich) for 5 min at room temperature and then mixed in 200 µl reaction buffer [4M GdnHCl (Sigma-Aldrich) and 10 µM ThT (Sigma-Aldrich) in phosphate buffered saline (PBS, pH7.4) (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea)] together with compounds (1–100 µM) and vehicle, DMSO. The PAFA reaction mixture was prepared in a 96-well black flat bottom polystyrene not treated microplate (Corning, Corning, NY, USA), sealed with a microplate adhesive film (USA Scientific, Ocala, FL, USA) and incubated at 37 °C for 60 h with shaking at 335 rpm in Infinite M200 Pro Fluorescence reader (Tecan, Maennedorf, Zurich, Switzerland). The fibrils formation was measured every 1 h by top reading of the fluorescence intensity using a 444 nm excitation and 485 nm emission filter.