Jul 26, 2024
Version 3
T cell purification and activation V.3
- Moustafa Nouh Elemeery1,2,
- Salix Boulet3,
- louis-eric.trudeau Trudeau4,
- Nathalie Labrecque5
- 1Department of neurosciences, Faculty of Medicine, Universite de Montreal, Canada;
- 2Medical Biotechnology Department, National Research Centre, Dokki, Cairo, Egypt;
- 3Centre de recherche de l’hôpital Maisonneuve-Rosemont (CRHMR), Department of Medicine, Faculty of Medicine, Université de Montréal, Canada;
- 4Department of Pharmacology and Physiology, Faculty of Medicine, Université de Montréal, Canada;
- 5Institut de recherches cliniques de Montréal (IRCM), Department of Microbiology, Infectious Diseases and Immunology, Faculty of Medicine, Université de Montréal, Canada
Protocol Citation: Moustafa Nouh Elemeery, Salix Boulet, louis-eric.trudeau Trudeau, Nathalie Labrecque 2024. T cell purification and activation. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbz431gpk/v3Version created by Lilia Rodriguez
Manuscript citation:
Elemeery, M. N., Tchung, A., Boulet, S., Mukherjee, S., Giguere, N., Daudelin, J. F., ... & Trudeau, L. E. (2024). Adoptive transfer of mitochondrial antigen-specific CD8+ T-cells in mice causes parkinsonism and compromises the dopamine system. bioRxiv, 2024-02.
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: July 26, 2024
Last Modified: July 26, 2024
Protocol Integer ID: 104171
Keywords: ASAPCRN, cell suspensions of splenocyte, activation of mouse naïve cd8, mouse spleen, cell suspension, splenocyte, removal with biotinylated antibody, unwanted cell, mouse naïve cd8, isolation kit from stemcell, labeled cell, cell, biotinylated antibody, purification kit, purification, stemcell
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP 000525
Disclaimer
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Abstract
This protocol details the purification and activation of Mouse Naïve CD8+ T Cell from mouse spleens using a purification kit. The cells are then activated by culturing them in plates coated with anti-CD3 antibodies and adding soluble anti-CD28 antibodies. The activation process occurs over 72 hours, with IL-2 added after the first 24 hours to support T cell proliferation and survival. The isolation kit from STEMCELL is designed to isolate naïve CD62L+CD44-CD8+ T cells from single-cell suspensions of splenocytes by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies that are directed against non-naïve CD8+ T cells (CD4, CD11b, CD11c, CD19, CD44, CD45R/B220, CD49b, TCRγ/δ, TER119) and streptavidin-coated magnetic particles. Labeled cells are separated using an EasySep‱ magnet without the use of columns.
Materials
Reagents and solution:
| A | B | C | |
| Reagent or solution | Supplier | Catalogue # | |
| Anti-mouse CD3 (Clone: 145-2C11) | Bioxcell | BE0001 | |
| Anti-mouse CD28 (clone: 37.51) | Leinco | C379-5.0 mg | |
| RPMI media | VWR (Corning) | CA45000-396 | |
| Phosphate Buffer saline (PBS) | Gibco | 14190144 | |
| EasySep buffer | STEMCELL | 20144 | |
| EasySep™ Mouse CD8+ T Cell Isolation Kit | STEMCELL | 19858 | |
| ACK (Ammonium-Chloride-Potassium) Lysing Buffer | ThermoFisher | A1049201 | |
| L-glutamine 200 mM | VWR | CA45000-676 | |
| HEPES 1M | Fisher | MT25060CI | |
| Sodium Pyruvate | VWR | CA45000-710 | |
| 2-Mercaptoethanol | ThermoFisher | 21985023 | |
| Non-Essential Amino acids | VWR (Corning) | CA45000-700 | |
| Fetal Bovin Serum | Gibco | 12483020 | |
| 24 wells plate flat bottom suspension plates | Sarstedt | 83.1836.500 | |
| 96 wells plate flat bottom suspension plates | Sarstedt | 82.1581.001 | |
| Cell strainers (70um) | Fisher | 08-771-2 |
InVivoMAb anti-mouse CD3BioXcellCatalog #BE0001
Anti-Mouse CD28 (Clone 37.51)Leinco Technologies Inc.Catalog #C379
RPMI VWR International (Avantor)Catalog #45000-396
Phosphate buffered saline (PBS) without Ca/Mg Thermo Fisher ScientificCatalog #14190144
EasySep™ BufferSTEMCELL Technologies Inc.Catalog #20144
EasySep™ Mouse Naïve CD8 T Cell Isolation KitSTEMCELL Technologies Inc.Catalog #19858
ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
L( )-Glutamine solution 200 mMVWR International (Avantor)Catalog #45000-676
Sodium pyruvate solution 100 mMVWR International (Avantor)Catalog #45000-710
2-mercaptoethanolGibco - Thermo Fisher ScientificCatalog #21985023
Non essential amino acidsVWR International (Avantor)Catalog #45000-700
Fetal Bovine Serum, qualified, CanadaThermo FisherCatalog #12483020
Falcon™ Cell StrainersFisher ScientificCatalog #08-771-2
RPMI complete (RPMIc):
| A | B | |
| RPMI | 500 mL | |
| FBS 10% | 50 mL (inactivated) | |
| L-Glutamine | 5 mL | |
| Sodium pyruvate | 5 mL | |
| Antibiotic (Pen-Strep) | 5 mL | |
| Non-essential amino acids | 5 mL | |
| HEPES | 5 mL | |
| 2-Mercaptoethanol | 50 mmol/L (final) |
Note
Note! Very important, 2-Mercaptoethanol is an essential growth factor for mouse T-lymphocytes.
Troubleshooting
Purification and activation
4d 0h 30m
Dilute CD3 antibody (145-2C11) (clone KT3 can also be used) to 1 µL in PBS.
Coat plates with anti-CD3 antibody.
Use 24 well (Sarstedt, cat# 83.1836.500) or 96 well (Sarstedt, cat# 82.1581.001) plates. If these plates are not used, there is a risk of partial stimulation due to low absorbance of the antibody on the plate.
Add 100 µL of antibody/well if 96 wells or 1 mL per well if 24 wells.
Incubate the plate for 24:00:00 at 4 °C or 01:00:00 at 37 °C .
1d
Remove the antibody (aspirate) and wash 2 times with PBS.
Add 100 µL of RPMIc if 96 wells or 1 mL if 24 wells and incubate the plate at 37 °C for 00:15:00 . See complete recipe for RPMIc in Materials section.
15m
Collect spleen from mice 6-8 weeks in complete RPMI media (RPMIc), sex matched with recipient mice.
Note: using spleens from 6-8 week old mice ensures that the isolated T cells are from young adult mice with a fully developed immune system, and the sex-matching helps prevent potential complications in downstream applications or experiments.
Purify CD8+ T cells using EasySep mouse naïve purification kit (STEMCELL, catalo # 19858) as follow:
Use a frosted microscope slide to homogenize spleens in PBS or Hanks' Balanced Salt Solution (HBSS) containing 2% fetal bovine serum (FBS).
Remove aggregates and debris by passing cell suspension through a 70 μm mesh nylon strainer. Collect cells in a 15 mL tube.
Centrifuge at 1300 rpm, 00:05:00 and discard the supernatant.
5m
Red blood cells lysis is done by adding5 mL /spleen of 0.83% ammonium chloride and incubate for 00:05:00 (or 00:02:00 for ACK lysing buffer) at Room temperature while continuously shaking tubes.
5m
Quench by filling up tube with RPMIc.
Centrifuge at 1300 rpm, 00:05:00 and discard supernatant.
5m
Resuspend in EasySep buffer at 1x108 cells/ml (Easysep mouse naïve CD8+ T cell isolation kit (#19858A) and follow the protocol provided by STEMCELL.
Wash cells with RPMIc medium.
Resuspend cells in 5 mL of RPMIc, count cells and adjust the concentration to 2x106 cells/mL
Add 100 µL of cells (2x105 cells) per well if 96 wells or 1 mL of cells (2x106 cells) per well if 24 wells).
Add purified anti-CD28 antibodies to reach a concentration of 5 µL .
Incubate the plate at 37 °C and 5% CO2 for 24:00:00 .
1d
Add 20 Unit/ml of IL-2.
Incubate the plate at 37 °C and 5% CO2 for another 48:00:00 .
2d
Note
*** Don't forget the non-stimulated controls.
- Resuspend the cells well throughout the experiment (the cells quickly settle to the bottom of the tube)
- Check purity of CD8+ T cells after STEMCELL isolation by staining with anti-CD8 antibody followed by flow cytometry.
- Check activation after stimulation using anti-CD8 and anti-CD44 staining followed by flow cytometry.