This prototypic method has evolved to use the lipid raft fractions of the plasma membrane as the source for PrPC because PrP conversion occurs at the caveolaelike membrane domains of neuronal cells. Recently, PrPC purified from brain tissue or cultured mammalian cells and recombinant PrP expressed in bacterial cells have replaced brain material for PMCA. Crude brain homogenate and the lipid raft fractions of the membrane provide a GDC-0879 905281-76-7 comprehensive set of components required for PMCA including a cofactor, while purified PrPC or recombinant PrP offers WZ4002 EGFR/HER2 inhibitor defined minimal substrates. However, availability of brain material from certain species or transgenic animals carrying the PrP gene with certain mutations and polymorphisms is often limited. Alternatively, preparation of the substrates by expression/purification of native PrPC from animal tissues and cell lines, as well as recombinant PrP from bacterial cells, requires additional, laborious steps. Thus, it is necessary to establish a convenient alternative that overcomes aforementioned drawbacks of the current PMCA method. In this study, we used cell lysate of cultured mammalian cell lines in PMCA reactions. Lysate of cultured cells has not been used as a substrate source for PMCA and it has been considered incapable of supporting PrPSc formation in PMCA unless complemented with brain homogenate that may include a cofactor for PrP conversion. Based on our recent observation that PrPC abundance is critical for robust PrPSc propagation in PMCA, we performed PMCA with PrP-expressing cell lysates in which the level of PrPC was equivalent to wild type brain material. Here, we show that PMCA replication of mouse and hamsteradapted PrPSc using cell lines that express murine and hamster PrPC, respectively. The current study demonstrates that cell lysate with concentrated PrPC allowed robust PMCA of PrPSc from multiple strains and species. The ability of cell lysate to support PrPSc formation in PMCA is comparable to that of wild type brain material. This result suggests that cell lysate can replace animal organ-derived material for in vitro PrPSc amplification without compromising PrP conversion efficiency.