A FRET Flow Cytometry Technique to Detect Tau-Seed Induced Reporter Protein Aggregation
Trascrizione
Take a mammalian cell culture expressing the repeat domain of the microtubule-binding protein tau.
These cytoplasmic tau reporters possess an aggregation-prone mutation and exist as monomers.
The reporters are fused to either fluorophore A or B, with the former's emission spectrum overlapping with the latter's absorption spectrum.
Add a liposome complexed with tau seeds or fibrillar aggregates of full-length tau.
Upon entering the cell, the liposome releases the seeds in the cytoplasm.
The released seeds mediate aggregation of the tau reporters, bringing fluorophore A near fluorophore B.
Add trypsin to detach the cells, then perform fixation.
Analyze the cells using a flow cytometer.
Due to the proximity within the aggregate, upon excitation, the energy transfer from fluorophore A to B via a dipole-dipole interaction is referred to as fluorescence resonance energy transfer or FRET.
The excited fluorophore B emits a FRET signal, the presence of which indicates protein aggregation.
