Jan 22, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.eq2lyjbbplx9/v1
  • Cole S Sitron1,
  • Victoria A Trinkaus1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Cole Sitron
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lyjbbplx9/v1
Protocol Citation: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl 2024. Induction of aggregation in alpha-synuclein-expressing cells by treatment with preformed fibrils (PFFs). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjbbplx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 16, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93747
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
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
957-2488.docx
16KB
Materials

Cell culture

Cells expressing alpha-synuclein

Reagents

  • Appropriate cell culture medium
  • ReagentLipofectamine™ 3000 Transfection ReagentThermo Fisher ScientificCatalog #L3000008
  • ReagentOpti-MEM™ Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
  • ReagentPBS, pH 7.2Thermo Fisher ScientificCatalog #20012068
  • Concentration5 mg/mL alpha-synuclein preformed fibrils (PFFs) (see dx.doi.org/10.17504/protocols.io.btynnpve for purification protocol)

Equipment

  • BioRuptor Plus sonicator (Diagenode cat. no. B01020001) (or equivalent)
Equipment
Bioruptor® Plus sonication device
NAME
Sonication device
TYPE
Bioruptor®
BRAND
B01020001
SKU
https://www.diagenode.com/en/p/bioruptor-plus-sonication-device
LINK







Induction of alpha synuclein aggregation
Induction of alpha synuclein aggregation
15m 10s
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 Temperature37 °C , thaw an aliquot of PFFs, and (if using) allow lipofectamine to reach TemperatureRoom 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 Amount100 µL and Amount300 µL for proper sonication in the Bioruptor Plus.


Pipetting
Sonicate the PFFs in the Bioruptor Plus on high for 25 cycles of Duration00:00:05 on and Duration00:00:05 off at Temperature4 °C .

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

10s
Make a master mix of PFF and PBS solutions.
Amount1 µL 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 Amount40 µL of sonicated & diluted PFFs.


Pipetting
Master mixes should contain a ratio of Amount50 µL OptiMEM : Amount20 µL sonicated & diluted PFFs (or PBS as a control) : Amount3 µL of lipofectamine 3000.

Note
For a 12-well plate, 100 OptiMEM : Amount40 µL sonicated & diluted PFFs/PBS : Amount6 µL lipofectamine 300 should be used.


Pipetting
If using lipofectamine, first incubate the lipofectamine 3000 in OptiMEM for Duration00:05:00 before adding PFFs or PBS. Gently vortex upon addition of lipofectamine 3000.

5m
Incubation
After addition of PFFs/PBS, gently vortex master mix solutions and incubate for Duration00:10:00 at TemperatureRoom temperature .

10m
Incubation
Add master mix solutions dropwise to cells, gently vortexing before adding to each well.
Pipetting
Protocol references

1. Luk KC, Song C, O'Brien P, Stieber A, Branch JR, Brunden KR, Trojanowski JQ, Lee VM. Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):20051-6. doi: 10.1073/pnas.0908005106. Epub 2009 Nov 5. PMID: 19892735; PMCID: PMC2785290.

2. Volpicelli-Daley LA, Luk KC, Lee VM. Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates. Nat Protoc. 2014 Sep;9(9):2135-46. doi: 10.1038/nprot.2014.143. Epub 2014 Aug 14. PMID: 25122523; PMCID: PMC4372899.