Fig. 6

Schematic representation of tau seeding assay utilising Tau RD P301S FRET biosensor cells – (A) If tau seeds propagate into these cells and initiate aggregation of the two tau constructs, CFP can be excited with a laser causing YFP emission due to the energy transfer of these being in close proximity. (B) Seeding can be monitored as punctate fluorescence using the GFP channel of a microscope (Scale bar, 20 μm) or by quantitative flow cytometry by measuring FRET response on a CFP vs. YFP emission bivariate plot (C) When specifically exciting CFP, cells can be termed FRET negative if they emit at 475 nm bandwidth or FRET positive if emission is at 530 nm. Figure adapted from [16, 26]. (D) Representative gating strategy and data analysis of AD brain seeding Tau RD P301S FRET biosensor cells with FlowJo (10.7) software with further details provided within Materials and methods section