Teflon beads

10% normal brain homogenate (NBH) from healthy hamsters or mice was prepared as described previously47 (link). To produce desialylated substrates for dsPMCAb, 10% NBH was treated with Arthrobacter ureafaciens sialidase (cat # P0722L, New England Biolabs, Ipswich, MA) as follows. After preclearance of NBH at 500 × g for 2 min and addition of the enzyme buffer supplied with the manufacturer to supernatant 200 units/mL of sialidase were added to the supernatant, incubated on a rotator at 37 °C for 5 h and the resulting material referred to as dsNBH substrate used in dsPMCAb.
PMCAb and dsPMCAb reactions were conducted as previously described18 (link)48 (link) using Misonix S-4000 microplate horn (Qsonica LLC, Newtown, CT) in the presence of two 2/32” Teflon beads in each tube (McMaster-Carr, Elmhurst, IL). One round consisted of 20 sec sonications delivered at 170 W energy output applied every 20 min during a 24 hour period. For each subsequent round, 10 μl (for hamster strains) or 20 μl (for mouse strains) of the reaction products from the previous round were mixed with 90 or 80 μl of fresh substrate, respectively, or as specified for the amplification rate experiment.

sPMCA was performed using one of two methods, as the assay has evolved over time in our laboratory. For both methods, a 10% normal brain homogenate (NBH) in 0.1 M PBS buffer (pH 7.5, with 1% Triton X-100) was prepared from whole brains collected from clinically healthy naïve transgenic mice (<4 months of age) that overexpress cervid PrP (TgCerPrP-E2265037) to serve as a substrate for the PrP^C to PrP^CWD seeded conversion reaction (prion seeding activity) in sPMCA as previously described [29 (link)]. Fetal tissue homogenate (30 μL; ranging from 2.5 to 20% w/v; Table 2) was added to 50 μL 10% w/v NBH and subjected to either sPMCA method 1 (run in 2013): seven 24 h-rounds of sonication with each round equaling 288 cycles of 10 s sonication/5 min incubation, or sPMCA method 2 (run in 2021): one round of 72 h (144 cycles) followed by four rounds of 24 h (48 cycles each) of 30 s sonication/29 min 30 s incubation. Both sPMCA methods were performed at 37 °C with Misonix sonicator setups. After each round, 30 μL amplified material was transferred to 0.2 mL PCR tubes containing 50 μL 10% NBH, two 2.38 mm and three 3.15 mm Teflon beads (McMaster-Carr, Elmhurst, IL, USA). sPMCA method 1 reactions were assessed by western blotting, whereas sPMCA method 2 reactions were assessed by RT-QuIC.