TITLE: Isolation of brain infiltrating lymphocytes
AUTHORS: Moustafa Nouh Elemeery, Salix Boulet, louis-eric.trudeau, Nathalie Labrecque

[DESCRIPTION]
This protocol details the isolation of brain infiltrating lymphocytes. Splenic lymphocytes are screened to verify the presence/phenotype of CD8 in the periphery. 

[STEPS]
SECTION: Infiltration procedure
1. Inject mice intravenously with CD45-FITC 3ug/mice (6 µL from vial/mice) for 3 min-5 min, then euthanized directly by decapitating head to stop blood circulation.

SECTION: Infiltration procedure
2. Collect the brain for each experimental condition vs control.

SECTION: Infiltration procedure
3. Dissociate brain in RPMIc plus collagenase-D 1 1524, DNase 150 ug/ml as follow:

SECTION: Infiltration procedure
1.1. CD45 is prepared as 180 µL CD45-FITC + 4320 µL PBS → to inject 150 ul/mice.

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1.2. Keep one mouse non injected for Live/Dead staining.
SECTION: Infiltration procedure
3.1. Place the brain in 15mm petri dish and add 2ml/brain of the collagenase-DNase mix.

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

SECTION: Infiltration procedure
3.3. Transfer to tube (gentleMACS TM C tubes, cat 130096334) and make sure to lock it.

SECTION: Infiltration procedure
3.4. Keep the brain pieces on the downside of the spiral bend.

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3.5. 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:
Temp. ON
Spin 200 rpm 1’’
Spin 300 rpm 1’’
Spin 400 rpm 3’’
Spin -400 rpm 3’’
Spin 400 rpm 3’’
Spin -400 rpm 1’’
Spin 200 rpm 4’’
Spin -400 rpm 2’’
Spin 400 rpm 3’’
Spin 200 rpm 6’’
Spin -200 rpm 2’’
Spin 200 rpm 8’’
Spin 0 rpm 45’ 0’’
Temp OFF

SECTION: Infiltration procedure
3.6. When dissociation finish (45 min), 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.

SECTION: Infiltration procedure
4. Centrifuge 2000 rpm, 5 min.

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

SECTION: Infiltration procedure
5.1. For 100 mL of 90% Percoll:
90 mLpercoll and 10 mL PBS 10X.

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5.2. For 45 mLof 70% Percoll:
35.7 mL from 90% Percoll + 10.3 mLPBS 1X.

SECTION: Infiltration procedure
5.3. For 43.75 mL of 37% Percoll:
18 mLfrom 90% Percoll +25.75 mLPBS 1X.

SECTION: Infiltration procedure
6. 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).

SECTION: Infiltration procedure
11. Centrifuge at  2000 rpm, 5 min.

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

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

SECTION: Infiltration procedure
8. After centrifugation, with the help of vacuum suction clear the upper lipid layer on the top of the tubes.

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7. Centrifuge 2000 rpm, 20 min, brake-off.

SECTION: Infiltration procedure
12. Stain 1x106 cells from the brain or 3x106 cells from the spleen with the live/dead stain and after with extracellular staining panel. 

 

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

SECTION: For splenic cell induction:
26. Add 100 µL cold 1X PBS per well.

SECTION: For splenic cell induction:
25. Wash in cold 1X PBS then repeat step 24.

SECTION: For splenic cell induction:
24. Centrifuge at 1300 rpm, 3 min, 12 °C and remove supernatant.

SECTION: For splenic cell induction:
23. Incubate the cells in presence of the re-stimulation mix for300 min at 37 °C and 5% CO2.

SECTION: For splenic cell induction:
22. Add 100 µL of the following restimulation mix (at 2X concentration). 

SECTION: For splenic cell induction:
21. Add 100 µL of cells (3 x 106 cells) per well in 96-Well Round-Bottom plate (Fisher # 07200760). 

SECTION: For splenic cell induction:
20. Add volume to reach 30x106 cells/ml.

SECTION: For splenic cell induction:
19. Count the cells on hemocytometer (1/100 dilution).

SECTION: For splenic cell induction:
18. Reconstitute in 2.5 mLof sterile RPMIc.

SECTION: For splenic cell induction:
17. Add 5 mL of sterile RPMIc and centrifuge at 1300 rpm, 5 min .

SECTION: For splenic cell induction:
16. Perform red blood cell lysis: 5 mL of NH4Cl 0.83%, 5 min at Room temperature.

SECTION: For splenic cell induction:
15. Centrifuge at 1300 rpm, 5 min.

SECTION: For splenic cell induction:
14. Harvest the spleen in the culture hood with frosted microscope slide. 

SECTION: For splenic cell induction:
22.1. Don’t forget to add one well of PMA-IONO control (2 µL of stock PMA 5 1527 and Ionomycine 50 1527) in 200 µL final. The final concentration is 50 1585 of PMA and 500 1585 Ionomycin.
 
SECTION: For splenic cell induction:
22.2. No-Stim (2X): RPMIc + 20 1527 Brefeldine A (BFA). 

SECTION: For splenic cell induction:
22.3. OVA Re-stimulation (2X): RPMIc + 20 1527 BFA final (stock solution at 1 1524) + 4 1527 OVA peptide. 

SECTION: For splenic cell induction:
22.4. SYRGL Restimulation (2X): RPMIc + 20 1527 BFA final (stock solution at 1 1524) + 4 1527 SYRGL peptide.

SECTION: For splenic cell induction:
22.5. For example:

for 4 mL of RPMIc, we add 1.6 µLof OVA peptide (stock at 10 1524) and 80 µLof BFA.
for 4 mL of RPMIc, we add 1.6 µL of SYRGL peptide (stock at 10 1524) and 80 µL of BFA (stock at 1 1524).

SECTION: For splenic cell induction:
31. Resuspend the cells in 200 µL of FACSWASH.

SECTION: For splenic cell induction:
30. Wash in cold 1X PBS (300 µL) and repeat step 24.

SECTION: For splenic cell induction:
29. Add 100 µL of 1X PBS and repeat step 24.

SECTION: For splenic cell induction:
28. Incubate 20 min atRoom temperature.

SECTION: For splenic cell induction:
27. Add 100 µL of formaldehyde 4% per well (up and down).

SECTION: For splenic cell induction:
32. 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):