Feb 03, 2025

Public workspaceα-synuclein PFF treatment

DOI
dx.doi.org/10.17504/protocols.io.q26g7m821gwz/v1
  • 1Institut Imagine;
  • 2ASAP
  • Team Deleidi
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.q26g7m821gwz/v1
Protocol Citation: Maria Jose Perez J., Federico Bertoli 2025. α-synuclein PFF treatment. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7m821gwz/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: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112831
Funders Acknowledgements:
ASAP
Abstract
α-synuclein PFF treatment
α-synuclein PFF treatment
α-synuclein PFF treatment
Shake Alpha-Synuclein Monomers in PBS for 7 days at 1,000 RPM. Solution should turn turbid during this period.
Spin fibrils for 10 minutes at max speed in benchtop centrifuge at Room temperature.
Discard supernatant and resuspend fibrils in PBS at a concentration of 5 ug/ul.
Validate α-synuclein PFFs via:
  • Electron microscopy
  • Sedimentation assay
  • Thioflavin-T assay (Cat. number T3516; Sigma‒Aldrich)
Label α-synuclein PFFs with an Alexa Fluor 594 kit (Cat. number A10239; Thermo Fisher Scientific) following the manufacturer’s instructions.
For microglial treatment:
For microglial treatment:
  • Dilute α-synuclein PFFs in PBS to 100 μg/mL.
  • Sonicate the solution (10-second pulses at 30% amplitude, six times with 2-minute intervals) using a Q700-220 sonicator (Qsonica, Newtown, CT, USA).
  • Dilute sonicated PFFs in microglial medium to a final concentration of 1 μg/μL.
Add α-synuclein PFFs to iPSC-derived microglia cultures and cotreat with CDDO-Me, etoposide, or MCHA as needed.
Fix cells with 4% PFA for 10 minutes after 30, 60, and 90 minutes of treatment.
Perform immunostaining as described in the paper.
Acquire images:
  • For immunostained samples: use a Leica TCS SP8 confocal microscope with a 60×/1.4 numerical aperture oil immersion objective.
  • For live-cell imaging: use an Evident APX100-HCU box-type microscope with a 20×/0.8 numerical aperture objective. Capture images every 20 minutes for 3 hours.
For quantitative analysis:
  • Select three random fields containing 5–15 cells per field.
  • Image and analyze these fields across 3 independent experiments.
Perform particle analysis using Speckle Counting and Object Tracking pipelines in CellProfiler Image Analysis Software (version 4.2.6, RRID: SCR_007358) according to recommended guidelines.
Detect cell bodies using CD68 staining or brightfield imaging.