Induction of aggregation in alpha-synuclein-expressing cells by treatment with preformed fibrils (PFFs)

Abstract

This protocol details how to efficiently produce alpha-synuclein aggregates in cells by templating the misfolding of intracellular alpha-synuclein through treatment with preformed fibrils of alpha-synuclein (PFFs).

Attachments

957-2488.docx

Steps

Induction of alpha synuclein aggregation

Seed cells into plates such that they are ~25% confluent the following day.

Note

For our HEK cells, the correct seeding density is ~250 cells/mm², which is equivalent to 100,000 cells in a 12-well dish.

On the following day, warm PBS and OptiMEM to 37°C, thaw an aliquot of PFFs, and (if using) allow lipofectamine to reach Room temperature.

Note

Lipofectamine greatly increases the ability of PFFs to nucleate intracellular alpha synuclein aggregation, perhaps by altering the endocytic route that PFFs use to enter the cell. It is therefore not recommended to use lipofectamine if the route of PFF entry is a concern in your experiment. Lipofectamine is additionally excessively toxic and should therefore be avoided in some cell types.

Dilute the thawed PFFs 1:20 into an eppendorf containing PBS.

Note

The final volume must be between 100µL and 300µL for proper sonication in the Bioruptor Plus.

Sonicate the PFFs in the Bioruptor Plus on high for 25 cycles of 0h 0m 5s on and 0h 0m 5s off at 4°C.

Note

Sonication breaks up the PFFs into smaller, more nucleation-competent fibrils.

Make a master mix of PFF and PBS solutions.

5.1.

1µL of sonicated & diluted PFFs should be added per 10 mm² of plate area (5 ug alpha-synuclein/10 mm²).

Note

For a 12-well plate, this would be 40µL of sonicated & diluted PFFs.

5.2.

Master mixes should contain a ratio of 50µL OptiMEM : 20µL sonicated & diluted PFFs (or PBS as a control) : 3µL of lipofectamine 3000.

Note

For a 12-well plate, 100 OptiMEM : 40µL sonicated & diluted PFFs/PBS : 6µL lipofectamine 300 should be used.

5.3.

If using lipofectamine, first incubate the lipofectamine 3000 in OptiMEM for 0h 5m 0s before adding PFFs or PBS. Gently vortex upon addition of lipofectamine 3000.

After addition of PFFs/PBS, gently vortex master mix solutions and incubate for 0h 10m 0s at Room temperature.

Add master mix solutions dropwise to cells, gently vortexing before adding to each well.

Abstract

Attachments

Steps

Induction of alpha synuclein aggregation

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