Jul 26, 2024

Public workspaceIsolation of brain infiltrating lymphocytes V.2

DOI
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: dx.doi.org/10.17504/protocols.io.dm6gpzo68lzp/v2
Protocol Citation: Moustafa Nouh Elemeery, Salix Boulet, louis-eric.trudeau 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 15, 2024
Last Modified: July 27, 2024
Protocol Integer ID: 104111
Keywords: ASAPCRN
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:

  • ReagentgentleMACS Octo Dissociator with HeatersMiltenyi BiotecCatalog # 130-096-427
  • ReagentCollagenase DMerck MilliporeSigma (Sigma-Aldrich)Catalog #11088882001
  • ReagentCorning® RPMI 1640, CorningVWR InternationalCatalog #45000-416
  • ReagentDeoxyribonuclease I from bovine pancreas, type IVMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5025
  • ReagentDulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo FischerCatalog #14190144
  • ReagentL(+)-Glutamine solution 200 mM in water (29.20 mg/ml) cell culture reagent, Corning®VWR InternationalCatalog #45000-676
  • ReagentCorning™ HEPES, LiquidFisher ScientificCatalog #MT25060CI
  • ReagentSodium pyruvate solution 100 mM with 8.5 g/L NaCl cell culture reagent, Corning®VWR InternationalCatalog #45000-710
  • Reagent2-MercaptoethanolThermo FisherCatalog #21985023
  • ReagentCorning® MEM (Minimum Essential Medium) Nonessential Amino Acids, CorningVWR InternationalCatalog #45000-700
  • ReagentFetal Bovine Serum, qualified, CanadaThermo FisherCatalog #12483020
  • ReagentACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
  • ReagentMicro test plate, 96 well, slip lid, flat bottom, PS, transparentSarstedtCatalog #82.1581.001
  • ReagentFalcon™ Cell StrainersFisher ScientificCatalog #08-771-2
  • ReagentPercollMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1644-500ML
  • ReagentPhorbol 12-myristate 13-acetate (PMA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1585
  • ReagentIonomycin from Streptomyces conglobatusMerck MilliporeSigma (Sigma-Aldrich)Catalog #I9657
  • ReagentBrefeldin 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 Amount1 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 Amount1 L .
  2. Filter sterile in 250 ml bottle.
  3. Keep at Temperature4 °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

  • ReagentCD101 Monoclonal Antibody (Moushi101), PE-Cyanine7, eBioscience™ThermofisherCatalog #25-1011-80
  • ReagentBrilliant Violet 711™ anti-mouse/human CD11b AntibodyBioLegendCatalog #101242
  • ReagentBrilliant Violet 421™ anti-mouse CD127 (IL-7Rα) AntibodyBioLegendCatalog #135027
  • ReagentAPC anti-mouse CD25 AntibodyBioLegendCatalog #102012
  • ReagentBrilliant Violet 605™ anti-mouse CD4 AntibodyBioLegendCatalog #100548
  • ReagentAPC/Cyanine7 anti-mouse/human CD44 AntibodyBioLegendCatalog #103028
  • ReagentFITC anti-mouse CD45.1 AntibodyBioLegendCatalog #110706
  • ReagentAlexa Fluor® 700 anti-mouse CD45.2 AntibodyBioLegendCatalog #109822
  • ReagentPerCP anti-mouse CD62L AntibodyBioLegendCatalog #104430
  • ReagentAPC anti-mouse CD69 AntibodyBioLegendCatalog #104513
  • ReagentBrilliant Violet 785™ anti-mouse CD8a AntibodyBioLegendCatalog #100750
  • ReagentPE anti-mouse CD183 (CXCR3) AntibodyBioLegendCatalog #126505
  • ReagentPE/Dazzle™ 594 anti-mouse CD186 (CXCR6) AntibodyBioLegendCatalog #151116
  • ReagentAPC anti-mouse/human KLRG1 (MAFA) AntibodyBioLegendCatalog #138412
  • ReagentPerCP/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
Infiltration procedure
1h 20m
1h 20m
Inject mice intravenously with CD45-FITC 3ug/mice (Amount6 µL from vial/mice) for Duration00:03:00 -Duration00:05:00 , then euthanized directly by decapitating head to stop blood circulation.

5m
CD45 is prepared as Amount180 µL CD45-FITC + Amount4320 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 Amount1 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 (Duration00:45:00 ), 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
Wash
Centrifuge Centrifigation2000 rpm, Room temperature, 00:05:00 .

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

For Amount100 mL of 90% Percoll:
  • Amount90 mL percoll and Amount10 mL PBS 10X.

For Amount45 mL of 70% Percoll:
  • Amount35.7 mL from 90% Percoll + Amount10.3 mL PBS 1X.

For Amount43.75 mL of 37% Percoll:
  • Amount18 mL from 90% Percoll +Amount25.75 mL PBS 1X.

Add Amount3 mL of 70% Percoll in a 15 ml tubes. Resuspend the brain pellet in Amount3 mL of 37% Percoll and overlay gently on top of 70% Percoll (drop on the wall to prevent disruption).

Pipetting
Centrifuge Centrifigation2000 rpm, 00:20:00 , brake-off.

20m
Centrifigation
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 toAmount10 mL with 1X PBS, gently invert the tube up and down at least one time.

Centrifuge at Centrifigation2000 rpm, 00:05:00 .

5m
Centrifigation
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:
For splenic cell induction:
5h 38m
5h 38m
Collect the spleen from CD45-FITC injected mice in Amount3 mL of sterile complete RPMI (RPMIc).

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

Centrifuge at Centrifigation1300 rpm, Room temperature, 00:05:00 .

5m
Centrifigation
Temperature
Perform red blood cell lysis: Amount5 mL of NH4Cl 0.83%, Duration00:05:00 at TemperatureRoom temperature .

5m
Temperature
Add Amount5 mL of sterile RPMIc and centrifuge at Centrifigation1300 rpm, Room temperature, 00:05:00 .

5m
Centrifigation
Pipetting
Temperature
Reconstitute in Amount2.5 mL of sterile RPMIc.

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

Add volume to reach 30x106 cells/ml.

Pipetting
Add Amount100 µL of cells (3 x 106 cells) per well in 96-Well Round-Bottom plate (Fisher # 07200760).

Add Amount100 µL of the following restimulation mix (at 2X concentration).

Pipetting
Don’t forget to add one well of PMA-IONO control (Amount2 µL of stock PMA Amount5 µL and Ionomycine Amount50 µL ) in Amount200 µL final. The final concentration is Amount50 µL of PMA and Amount500 µL Ionomycin.
No-Stim (2X): RPMIc + Amount20 µL Brefeldine A (BFA).

OVA Re-stimulation (2X): RPMIc + Amount20 µL BFA final (stock solution at Amount1 µL ) + Amount4 µL OVA peptide.

SYRGL Restimulation (2X): RPMIc + Amount20 µL BFA final (stock solution at Amount1 µL ) + Amount4 µL SYRGL peptide.

For example:

  • for Amount4 mL of RPMIc, we add Amount1.6 µL of OVA peptide (stock at Amount10 µL ) and Amount80 µL of BFA.
  • for Amount4 mL of RPMIc, we add Amount1.6 µL of SYRGL peptide (stock at Amount10 µL ) and Amount80 µL of BFA (stock at Amount1 µL ).

Incubate the cells in presence of the re-stimulation mix forDuration05:00:00 at Temperature37 °C and 5% CO2.

5h
Incubation
Centrifuge at Centrifigation1300 rpm, 12°C, 00:03:00 and remove supernatant.

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

Wash
Add Amount100 µL cold 1X PBS per well.

Pipetting
Add Amount100 µL of formaldehyde 4% per well (up and down).

Pipetting
Incubate Duration00:20:00 atTemperatureRoom temperature .

20m
Incubation
Temperature
Add Amount100 µL of 1X PBS and repeat step 24.

Pipetting
Wash in cold 1X PBS (Amount300 µL ) and repeat step 24.

Wash
Resuspend the cells in Amount200 µL of FACSWASH.

Keep at Temperature4 °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