Fig. 4

Novel biosensor HEK293T clone 18 amplifies bioactive tau seeds with high sensitivity and specificity. A. Brain extracts from AD cases extensively characterized in Dujardin et al. [15], considered as low, moderate, and high seeing activity were tested in flow cytometry-based seeding assays in both the novel and original biosensor lines. Signal was normalized on the high seeder to demonstrate a similar pattern in both lines. Bars represent mean of 2 individual independent experiments. B. The specificity of the new tau biosensor clone was confirmed after immunodepleting of tau in AD brain homogenate. Tau was immunodepleted from brain lysate and both input and depleted material were tested in flow-cytometry based seeding assay. Immunodepletion was confirmed by western blot using a polyclonal tau antibody as detection (C): lane 1 is input, lane 2 is immunodepleted sample. Membrane shown on the right is a long exposure time showing a smear corresponding to High molecular weight (HMW) tau. D. Comparison of tau seeding activity in both biosensor lines across various tauopathies (each point represents the mean of 3 technical replicates of one case, error bars represent the standard deviation). E. A stable CHO clone overexpressing the biosensor construct was incubated with AD brain-derived HMW seeding competent tau or low molecular weight (LMW) tau in absence of liposomes for 48 h and uptake and amplification of seeds was quantified by flow cytometry. The addition or 1 μM RAP, a known LRP-1 inhibitor decreased seeding