Apr 08, 2025

Public workspaceDifferential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - CELSR2 staining protocol

DOI
dx.doi.org/10.17504/protocols.io.rm7vzk934vx1/v1
  • 1La Jolla Institute for Immunology
Emil Johansson
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzk934vx1/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 - CELSR2 staining protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzk934vx1/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 08, 2025
Protocol Integer ID: 119902
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000375
Abstract
The purpose of this protocol is to quantify the number of CD4 T cells expressing CELSR2.
Prepare FACS buffer
Prepare FACS buffer
FACS buffer recipe (2mM EDTA, 2%FBS in PBS)
  1. 500ml PBS pH 7.4 Gibco
  2. 10 ml FBS
  3. 2ml 0.5 M EDTA
  4. Filter through 0.2µm filter and de-gas for 30 minutes
  5. Store at 4˚C
Setup

  1. Thaw indicated number of vial(s) of PBMC.
  2. Place the vials in 37C water bath for 1 min. Transfer to sterile 15 ml Falcon Tubes with 10 mL HR5 and 20 µL benzonase.
  3. Centrifuge @ 1200 rpm (9/9 accel/break) for 7 min @ 4C.
  4. Resuspend cells in 10 ml HR5 and count cell number.
  5. Centrifuge cells @ 1200 rpm for 7 min @ 4C.
  6. Resuspend HR5 medium to get a final concentration of 10x106 cells/ml.
  7. Seed 100 µl of the 10x106 cells/ml cell suspension into 96U well plate (106 cells/100uL) for antibody staining.
Antibody staining

  1. Centrifuge cells at 1400 rpm/5 min/4C.
  2. Wash with 200 uL PBS per well and cfg at 1400 rpm/5 min/4C. ]
  3. Add 100µl/pts of Live/Dead eF506 (1/1000 in PBS) and Fc Block (1/80 in PBS). Incubate 20 min in dark at 4C.

ReagentFlurochromeAmount per well (100 uL total)
Viability DyeeF5060.1
Fc Block-1.25
PBS-98.65

  1. Add 100uL of FACS Buffer/well. Centrifuge cells at 1400 rpm/5 min/4C.
  2. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  3. Resuspend cells in 50 uL of antibody mix and incubate for 30 min at 4C.


Antibody targetFlurochromeAmount per well (100 uL total)
CD8aBV6501.25
CD19PE-Cy71.25
CCR7PerCP-Cy5.51.25
CD45RAeF4502.5
CD3AF7002.5
CD4BV7111
CELSR2Unconjugated0.5
Brilliant stain buffer plus-10
FACS Buffer-79.75


  1. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  2. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  3. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  4. Resuspend in 100 µL secondary antibody mix (99.6 µl FACS buffern and 0.4 µl PE- F(ab')2-Goat anti-Rabbit IgG (H+L) (A10542) per well) and incubate for 30 min at 4C.
  5. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  6. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  7. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  8. Resuspend in 150 µL of FACS Buffer /well.
  9. Ready to acquire on flow cytometer