Apr 22, 2025

Public workspaceDifferential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - FACS of CELR2 expressing or non-expressing cells

DOI
dx.doi.org/10.17504/protocols.io.q26g7mn48gwz/v1
  • 1La Jolla Institute for Immunology
Emil Johansson
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DOI: dx.doi.org/10.17504/protocols.io.q26g7mn48gwz/v1
Protocol Citation: Antoine Freuchet, Emil Johansson, cecilia, Alessandro Sette 2025. Differential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - FACS of CELR2 expressing or non-expressing cells. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mn48gwz/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: February 11, 2025
Last Modified: April 22, 2025
Protocol Integer ID: 119907
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000375
Abstract
The purpose of this protocol is to sort CD4 Tem cells either expressing or not expressing CELSR2, to allow subsequent scRNAseq analysis.
Differential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - FACS sorting
Differential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - FACS sorting
Setup
Thaw vial(s) in waterbath for ~1min30sec
Transfer into warm 10mL HR5 + 20uL of Benzonase. Centrifuge 400g, 5min, RT
Resuspend in 10mL of PBS-BSA 0.04% and count. Centrifuge 400g, 5min, RT
Resuspend at 20.10^6 cells/mL in PBS-BSA 1% (chilled). Add 1 ml
Staining
  1. Centrifuge 400g, 5min, RT.
  2. incubate cells with Fc blocking solution (90 µl PBS and 10 µl FC block) for 10 min at 4C.
  3. Centrifuge 400g, 5min, RT.
  4. Resuspend cells in staining solution. ubcubate for 30 in at 4C in the dark
Antibody targetFlurochromeAmount per well (100 µl per well)
CD3AF7002.5
CD45RAPE-Cy72.5
CD8aBV6501.25
CCR7PerCP-Cy5.51.25
CD4BV7111
CELSR2Purified0.25
HASHTAG-1
BV Buffer-10
PBS-BSA 1% -80.25

  1. Centrifuge 400g, 5min, RT.
  2. Wash three times in PBS-BSA 1%.
  3. Resuspend in 2 mL secondary antibody mix (1992 µl PBS-BSA 1% and 8 µl PE- F(ab')2-Goat anti-Rabbit IgG (H+L) (A10542) per sample) and incubate for 30 min at 4C.
  4. Wash three times in PBS-BSA 1%.
  5. Resuspend in 2 mL of PBS-BSA 1%. Add DAPI prior to sorting to stain dead cells
  6. Sort in 200 000 cells into PBS-FBS 20% pre-coated tubes