Prion Amplification from Animal Brains

Experiments using PMCA were performed as previously described [75 (link)]. Briefly, HY TME, DY TME or 139H-infected brains were homogenized to 10% w/v in DPBS (Mediatech, Herndon, VA) or enriched PrP^Sc was used as a PMCA seed. Uninfected brain and spleen tissues were homogenized to 10% w/v and 20% w/v in conversion buffer respectively, and used as a PMCA substrate. PMCA seeds were diluted in PMCA substrate at a 1:100 ratio e.g. 1 μl of seed was diluted into 99 μl of substrate. PMCA was performed with a Misonix 4000 sonicator (Farmingdale, NY). For PMCA with uninfected brain as substrate the sonicator output set to level 75 and an average power output of 160 watts during each sonication cycle. For PMCA with uninfected spleen as substrate, the sonicator output was set to level 87 with an average power output of 220 watts during each sonication cycle. One round of PMCA consist of 144 cycles; one cycle comprises of five-second sonication followed by a ten-minute incubation at 37°C. All PMCA sample groups had an n = 3 and all experiments were replicated a minimum of three times.