Model s3000

PMCA was performed as previously described [51 (link)]. Briefly, as a substrate, we used brain specimens obtained from outbred CD1 animals. Brains were harvested after perfusion, and 10% homogenate was prepared in conversion buffer (PBS containing 150 mM sodium chloride and 1% Triton X-100) with the addition of protease inhibitors. Ten μl of raw fibril preprations, cell lysates from infected cells (P6) or brain homogenates from infected mice were added to the substrate and transferred in 0.2 mL tubes, positioned on an adaptor placed on the plate holder of a microsonicator (Misonix, Model S3000) and subjected to 96 cycles of PMCA. Each cycle (also referred as PMCA round) consisted of 29 min and 40 sec of incubation at 37/40°C followed by a 20 sec pulse of sonication set at potency of 260–270 Watt. After one round of PMCA, an aliquot of the amplified material was diluted 10-folds into fresh substrate and a further PMCA run performed following the same procedure. To increase PMCA efficiency, teflon beads (n = 3) were added to the samples before each round of amplification. To avoid samples cross-contamination between each round, thorough decontamination of instruments and equipment was performed using 2N sodium hydroxide (NaOH) or 4M guanidine hydrochloride (Gdn-HCl).