A FRET Flow Cytometry Technique to Detect Tau-Seed Induced Reporter Protein Aggregation

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.

To replate the biosensor cells under sterile conditions, rinse each cell line with warm PBS followed by a 3-minute incubation with Trypsin-EDTA. When the cells have detached, stop the reaction with warm culture medium, and immediately transfer the cells into a conical tube.

Centrifuge the cells, resuspending the pellet in fresh medium. Then, count the cells and make a mastermix cell dilution of 3,500,000 cells per 13 milliliters of medium. Using a multichannel pipette, slowly transfer 130 microliters of the cells into each well of a flat-bottom tissue culture-treated 96-well plate taking care to place the pipette tips in the center of the wells without touching the bottom of the plate.

Once the cells have been plated, allow them to settle, undisturbed for 10 minutes at room temperature, and then incubate the plate overnight at 37 degree Celsius, 5% carbon dioxide, and greater than or equal to 80% relative humidity. The next day when the biosensor cells are 60% to 65% confluent, combine reduced serum medium, liposome reagent, and biological seed source to make transduction complexes.

Then, gently pipette 20 microliters of transduction complex down the side of each individual biosensor well and return the treated cells to the incubator for another 24 to 48 hours. Before harvesting, untreated cells will show only diffused fluorescence. However, cells treated with tau seed material will show bright aggregates after 1 to 2 days.

To harvest the cells, replace the cell culture medium with 50 microliters of Trypsin-EDTA, stopping the reaction after 5 minutes with 150 microliters of chilled culture medium. Immediately, transfer the cells to a 96-well round-bottom plate and pellet the cells. Aspirate the supernatant without disturbing the pellets, and then gently but thoroughly resuspend the cells in 50 microliters of 2% paraformaldehyde for 10 minutes. Then, spin down the cells again and resuspend the pellets in 200 microliters of chilled flow cytometry buffer.