Seeding assays in HEK cells
A cell-based seeding assay takes the question out of the tube. Instead of asking whether a protein forms fibrils in buffer, it asks whether material from a tube or from tissue can induce aggregation of that protein inside a living cell.
The standard system uses HEK293 cells stably expressing an aggregation-prone fragment of the protein of interest fused to a pair of fluorescent proteins that form a FRET pair. For tau, the widely used biosensor line expresses the repeat domain carrying the P301S mutation, fused separately to cyan and yellow fluorescent proteins. Alone, the fusion stays diffuse and FRET is low. When seeds enter the cell and template aggregation, the reporters come together, FRET rises, and visible puncta form.
Two readouts, one assay
FRET by flow cytometry gives a quantitative, population-level number: the fraction of cells with FRET and the intensity of the signal, usually combined into an integrated measure. It handles many conditions at once and is the right readout for ranking.
Puncta by imaging shows the aggregates directly, gives morphology, and catches artifacts that flow cannot. It is slower and it is the defensible way to confirm that a FRET signal is really aggregation.
Running both on the same experiment is worth the extra effort when a result is going to matter.
Getting seeds in
Seeds do not cross the membrane efficiently on their own, so they are usually delivered with a lipid transfection reagent. That step is also the assay's biggest source of variability. Keep the lipid to seed ratio fixed, prepare complexes the same way every time, and include a lipid-only control, because the reagent alone is mildly toxic and can produce background.
Seed preparation matters as much. Sonication changes fibril length and seeding potency, so a defined sonication step belongs in the protocol. Recombinant fibrils, brain-derived material and material from a previous cell passage are not interchangeable, and the choice changes what the assay is testing.
Testing an antibody
The usual design preincubates the seeds with the antibody before delivery, then asks whether seeding is reduced. It measures whether the antibody can neutralize seeds, which is the mechanism most extracellular anti-aggregation antibodies are proposed to work through.
Controls that decide whether the result is real:
- An isotype control at the same concentration, since the lipid complex is sensitive to added protein.
- A titration, not a single concentration, so partial effects are visible and a dose response can be shown.
- A no-seed arm, to establish the floor.
- A toxicity check, since a drop in FRET-positive cells can come from fewer living cells rather than less seeding.
- A reference antibody with known behavior, where one exists.
What it does and does not tell you
It tells you that material is biologically active in a cell, and that an antibody does or does not neutralize it under these conditions. That is much closer to disease-relevant activity than a ThT curve.
It does not tell you what happens in a neuron, in tissue or in a brain. The biosensor line overexpresses a fragment, the seeds are delivered artificially, and neither reflects uptake and propagation in vivo. Treat it as the strongest cell-level evidence available in this area, and say what it is.
How we run it
We run seeding in biosensor lines with matched isotype, lipid-only and no-seed controls, read FRET for ranking and confirm by imaging, and pair the result with ThT kinetics so mechanism and activity are measured separately.