Jul 26, 2024

Isolation of brain infiltrating lymphocytes V.2

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpzo68lzp/v2
  • 1Department of Neurosciences, Faculty of Medicine, Université de Montréal, Canada;
  • 2Medical Biotechnology Department, National Research Centre, Dokki, Cairo, Egypt;
  • 3Centre de recherche de l’hôpital Maisonneuve-Rosemont (CRHMR), 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
Lilia Rodriguez
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpzo68lzp/v2
Protocol Citation: Moustafa Nouh Elemeery, Salix Boulet, louis-eric.trudeau , Nathalie Labrecque 2024. Isolation of brain infiltrating lymphocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzo68lzp/v2Version 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 27, 2024
Protocol  Integer ID: 104111
Keywords: ASAPCRN, brain infiltrating lymphocyte, isolation of brain infiltrating, splenic lymphocyte, brain infiltrating, phenotype of cd8, isolation, cd8, protocol details the isolation
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP 000525
Disclaimer
ETHICS DISCLAIMER

The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol details the isolation of brain infiltrating lymphocytes. Splenic lymphocytes are screened to verify the presence/phenotype of CD8 in the periphery.
Materials
Materials and reagents:

  • gentleMACS Octo Dissociator with HeatersMiltenyi BiotecCatalog # 130-096-427
  • Collagenase DMerck MilliporeSigma (Sigma-Aldrich)Catalog #11088882001
  • Corning® RPMI 1640, CorningVWR International (Avantor)Catalog #45000-416
  • Deoxyribonuclease I from bovine pancreas, type IVMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5025
  • Dulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo Fisher ScientificCatalog #14190144
  • L( )-Glutamine solution 200 mM in water (29.20 mg/ml) cell culture reagent, Corning®VWR International (Avantor)Catalog #45000-676
  • Corning™ HEPES, LiquidFisher ScientificCatalog #MT25060CI
  • Sodium pyruvate solution 100 mM with 8.5 g/L NaCl cell culture reagent, Corning®VWR International (Avantor)Catalog #45000-710
  • 2-MercaptoethanolThermo FisherCatalog #21985023
  • Corning® MEM (Minimum Essential Medium) Nonessential Amino Acids, CorningVWR International (Avantor)Catalog #45000-700
  • Fetal Bovine Serum, qualified, CanadaThermo FisherCatalog #12483020
  • ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
  • Micro test plate, 96 well, slip lid, flat bottom, PS, transparentSarstedtCatalog #82.1581.001
  • Falcon™ Cell StrainersFisher ScientificCatalog #08-771-2
  • PercollMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1644-500ML
  • Phorbol 12-myristate 13-acetate (PMA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1585
  • Ionomycin from Streptomyces conglobatusMerck MilliporeSigma (Sigma-Aldrich)Catalog #I9657
  • Brefeldin AMerck MilliporeSigma (Sigma-Aldrich)Catalog #B6542-5MG

RPMIc:
RPMI (500 mL)
FBS 10 % (50 mL decomplemented)
L-Glutamine (5 mL)
Sodium pyruvate (5 mL)
HEPES (5 mL)
Antibiotic (Pen-Strep) (5 mL)
Non-essential amino acids (5 mL)
HEPES buffer (5ml)
b-mercaptoethanol 50 mmol/L final (Very important, essential growth factor for mouse T-lymphocytes).

  • 4% formaldehyde:
AB
PBS 10X14 ml
Formaldehyde 37.5%10.8 ml
Distilled H2O75.2 ml
Filter through 0.2 µm
Keep at 4°C.

FACSWASH:
  • For 1 L volume Mix:
AB
DMEM without phenol-red in powder10 g
Horse serum30 mL
HEPES 1M 30 mL
sodium azide 10% 10 mL
  1. Dissolve the mix in H2O mQ and complete to 1 L .
  2. Filter sterile in 250 ml bottle.
  3. Keep at 4 °C .

List of Antibodies:
ABCD
AntibodySupplierCatalogue #
rat anti-mouse CD8 (1B2)BiotinCustom madeF23.1, 53-6.72
CD101 (Moushi101)PE-Cy7ThermoFisher25-1011-80
CD11b (M1/70)BV711Biolegend101242
CD127 (A7R34)BV421Biolegend135027
CD25 (PC61)APCBiolegend102012
CD4 (RM4-5)BV605Biolegend100548
CD44 (IM7)APC-cy7Biolegend103028
CD45.2 (104)FITCBiolegend110706
CD45.2 (104)Alexa flour 700Biolegend109822
CD62L (MEL)PercPBiolegend104430
CD69 (H1.2F3)APCBiolegend104513
CD8 (53-6.7)BV785Biolegend100750
CXCR3 (CXCR3-173)PEBiolegend126505
CXCR6 (SA051D1)PEdazzle594Biolegend151116
KLRG1 (1MAFA)APCBiolegend138412
P2XR7 (1F11)PercP-Cy5.5Biolegend148710

  • CD101 Monoclonal Antibody (Moushi101), PE-Cyanine7, eBioscience™ThermofisherCatalog #25-1011-80
  • Brilliant Violet 711™ anti-mouse/human CD11b AntibodyBioLegendCatalog #101242
  • Brilliant Violet 421™ anti-mouse CD127 (IL-7Rα) AntibodyBioLegendCatalog #135027
  • APC anti-mouse CD25 AntibodyBioLegendCatalog #102012
  • Brilliant Violet 605™ anti-mouse CD4 AntibodyBioLegendCatalog #100548
  • APC/Cyanine7 anti-mouse/human CD44 AntibodyBioLegendCatalog #103028
  • FITC anti-mouse CD45.1 AntibodyBioLegendCatalog #110706
  • Alexa Fluor® 700 anti-mouse CD45.2 AntibodyBioLegendCatalog #109822
  • PerCP anti-mouse CD62L AntibodyBioLegendCatalog #104430
  • APC anti-mouse CD69 AntibodyBioLegendCatalog #104513
  • Brilliant Violet 785™ anti-mouse CD8a AntibodyBioLegendCatalog #100750
  • PE anti-mouse CD183 (CXCR3) AntibodyBioLegendCatalog #126505
  • PE/Dazzle™ 594 anti-mouse CD186 (CXCR6) AntibodyBioLegendCatalog #151116
  • APC anti-mouse/human KLRG1 (MAFA) AntibodyBioLegendCatalog #138412
  • PerCP/Cyanine5.5 anti-mouse P2X7R AntibodyBioLegendCatalog #108710


Brain infiltrating T cells staining panel:
  • ZNIR-APC/Cy7
  • CD8a-BV785
  • CD44-BV650
  • 1B2 -PE
Or Tet_OVA
  • CD69-APC
  • CD62L-BV421
  • P2X7-PercP-Cy5.5
  • CD101-PE-Cy7
  • CXCR3-BV510
  • CXCR6-PE-dazzle
  • CD11-BV711
  • CD45.2-APC-eF780
  • CD4-BV605




Infiltration procedure
1h 20m
Inject mice intravenously with CD45-FITC 3ug/mice (6 µL from vial/mice) for 00:03:00 -00:05:00 , then euthanized directly by decapitating head to stop blood circulation.

5m
CD45 is prepared as 180 µL CD45-FITC + 4320 µL PBS → to inject 150 ul/mice.

Keep one mouse non injected for Live/Dead staining.
Collect the brain for each experimental condition vs control.

Dissociate brain in RPMIc plus collagenase-D 1 µL , DNase 150 ug/ml as follow:

Place the brain in 15mm petri dish and add 2ml/brain of the collagenase-DNase mix.

Cut the brain into 8 longitudinal pieces and collect in one tube using 1ml tip (cut the tip to allow pieces to go through).

Transfer to tube (gentleMACS TM C tubes, cat 130096334) and make sure to lock it.

Keep the brain pieces on the downside of the spiral bend.

Fix the tube to the machine and select the brain dissociation (preset the machine as shown below in the brain dissociation machine setup section)→OK, will turn light and start homogenization.

Tissue dissociation machine setup:
  1. Temp. ON
  2. Spin 200 rpm 1’’
  3. Spin 300 rpm 1’’
  4. Spin 400 rpm 3’’
  5. Spin -400 rpm 3’’
  6. Spin 400 rpm 3’’
  7. Spin -400 rpm 1’’
  8. Spin 200 rpm 4’’
  9. Spin -400 rpm 2’’
  10. Spin 400 rpm 3’’
  11. Spin 200 rpm 6’’
  12. Spin -200 rpm 2’’
  13. Spin 200 rpm 8’’
  14. Spin 0 rpm 45’ 0’’
Temp OFF

When dissociation finish (00:45:00 ), homogenize with the end of the 5ml syringe over 70 um strainer on a 50 ml falcon tube. Wash the strainer with PBS to ensure the collection of all cells.

45m
Centrifuge 2000 rpm, Room temperature, 00:05:00 .

5m
During that time prepare 37% and 70% percoll from 90% percoll in 10X PBS.

For 100 mL of 90% Percoll:
  • 90 mL percoll and 10 mL PBS 10X.

For 45 mL of 70% Percoll:
  • 35.7 mL from 90% Percoll + 10.3 mL PBS 1X.

For 43.75 mL of 37% Percoll:
  • 18 mL from 90% Percoll +25.75 mL PBS 1X.

Add 3 mL of 70% Percoll in a 15 ml tubes. Resuspend the brain pellet in 3 mL of 37% Percoll and overlay gently on top of 70% Percoll (drop on the wall to prevent disruption).

Centrifuge 2000 rpm, 00:20:00 , brake-off.

20m
After centrifugation, with the help of vacuum suction clear the upper lipid layer on the top of the tubes.

Transfer the intermediate layer containing the cells to a new labelled 15ml tube.

Complete the volume to10 mL with 1X PBS, gently invert the tube up and down at least one time.

Centrifuge at 2000 rpm, 00:05:00 .

5m
Stain 1x106 cells from the brain or 3x106 cells from the spleen with the live/dead stain and after with extracellular staining panel.

Note
Do not forget to add a compensation for FITC in the compensation beads.

For splenic cell induction:
5h 38m
Collect the spleen from CD45-FITC injected mice in 3 mL of sterile complete RPMI (RPMIc).

Harvest the spleen in the culture hood with frosted microscope slide.

Centrifuge at 1300 rpm, Room temperature, 00:05:00 .

5m
Perform red blood cell lysis: 5 mL of NH4Cl 0.83%, 00:05:00 at Room temperature .

5m
Add 5 mL of sterile RPMIc and centrifuge at 1300 rpm, Room temperature, 00:05:00 .

5m
Reconstitute in 2.5 mL of sterile RPMIc.

Count the cells on hemocytometer (1/100 dilution).

Add volume to reach 30x106 cells/ml.

Add 100 µL of cells (3 x 106 cells) per well in 96-Well Round-Bottom plate (Fisher # 07200760).

Add 100 µL of the following restimulation mix (at 2X concentration).

Don’t forget to add one well of PMA-IONO control (2 µL of stock PMA 5 µL and Ionomycine 50 µL ) in 200 µL final. The final concentration is 50 µL of PMA and 500 µL Ionomycin.
No-Stim (2X): RPMIc + 20 µL Brefeldine A (BFA).

OVA Re-stimulation (2X): RPMIc + 20 µL BFA final (stock solution at 1 µL ) + 4 µL OVA peptide.

SYRGL Restimulation (2X): RPMIc + 20 µL BFA final (stock solution at 1 µL ) + 4 µL SYRGL peptide.

For example:

  • for 4 mL of RPMIc, we add 1.6 µL of OVA peptide (stock at 10 µL ) and 80 µL of BFA.
  • for 4 mL of RPMIc, we add 1.6 µL of SYRGL peptide (stock at 10 µL ) and 80 µL of BFA (stock at 1 µL ).

Incubate the cells in presence of the re-stimulation mix for05:00:00 at 37 °C and 5% CO2.

5h
Centrifuge at 1300 rpm, 12°C, 00:03:00 and remove supernatant.

3m
Wash in cold 1X PBS then repeat step 24.

Add 100 µL cold 1X PBS per well.

Add 100 µL of formaldehyde 4% per well (up and down).

Incubate 00:20:00 atRoom temperature .

20m
Add 100 µL of 1X PBS and repeat step 24.

Wash in cold 1X PBS (300 µL ) and repeat step 24.

Resuspend the cells in 200 µL of FACSWASH.

Keep at 4 °C in parafilm and perform the intracellular staining within 5 days (it is better to do it the day after).

Plate layout_2C (2 plates; Intracellular staining and Isotypic control):
ABCDEFGHIJK
2C_WT_male PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
2C_WT_male PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
2C_WT_female PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
2C_WT_female PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
PTx_CTL PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
Plate layout_OT1(2 plates; Intracellular staining and Isotypic control):
OT1_WT PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
OT1_KO PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated
PTx_CTL PMA/IONO/BFA SYRGL SIINFEKL BFA Non-treated