TITLE: Brain infiltrating leukocytes (BILs) extraction
AUTHORS: Alexandra Kazanova, Nathalia Oliveira, samantha.gruenheid

[DESCRIPTION]
This protocol details the extraction of brain infiltrating leukocytes (BILs).

[STEPS]
SECTION: Procedure (work as fast and as gentle as possible):
1. Prior to sacking the mice, prepare 15 mL conical tubes with 3 mL RPMI and weigh each tube.
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2. Inject mice with 200 µL of aCD45-FITC solution IP.

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3. Sac mice exactly at 20 min post injection by cervical dislocation.

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4. Carefully collect the brain with spatula to the 15 ml conical tube containing 3 ml RPMI and place on ice.

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5. Weigh tube to calculate the weight of the brains. 
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6. Add brain into the petri dish, cut in small pieces with scissors.
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7. Collect the brain pieces back to the collection 15 ml tube using a cut 1mL tip.
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8. Spin down the contents at 1500 rpm, 5 min, 4 °C. While spinning prepare Digestion enzyme mix. 
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9. Add 3 mL of RT digestion enzyme mix (1x) to each tube, mix gently (can be done using the vortex).

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10. Incubate the tubes at 37 °C 35 min with gentle shaking at 15 min and 30 min.
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11. After the digestion, put the tube immediately on ice. Add 5 mL of PBS (with 2mM EDTA and 10% FBS) to neutralize the enzymes.

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12. Prime cell strainers on top of the 50 ml tube with 10 mL FACS buffer.

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13. Add the digested brain through the strainer. Add another 5 mL FACS to flush the strainer. 

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14. Add digested brain and push it with 3ml syringe plunger through the strainer, keep pouring FACS buffer to flush the strainer and the tube for digestion (up to 50mL). 
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15. Spin cells at 300 x g, 5 min. 

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16. Prepare RT gradient (For each brain prepare 3.7 mL of Percoll and 5.3 mL D-PBS containing calcium and magnesium).  

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17. Discard supernatant after centrifugation and resuspend very gently the cell pellet in 1 mL of D-PBS. Transfer the pellet to the 15ml conical tube containing 9 mL of gradient solution. (Total gradient solution = 10mL). 

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18. Centrifuge at 500 x g, 20 min No Brake. 

 

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19. Collect and add the upper laye of myelin, using the cut 1 ml tips.


 

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21. Add ice cold PBS to the top and spin down at400 x g, 5 min, 4 °Cwith brake. Discard the supernatant.

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20. Collect the interphase up to the lowest red cell containing sediment and transfer it to the 50 ml tube (around 7mL).
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22. If any of the pellets are contaminated with RBC/debris then: 

Add 1 mL ACK lysis buffer and mix with the pipette and leave for for 30 s and block the buffer with 4 mL of 10% FBS in PBS. Spin down at 400 x g, 5 min, 4 °C with brake. Discard the supernatant.

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23. Resuspend the pellet in 200 µL of FACS buffer, take a small aliquot to stain trypan blue and count (from a UI mouse you expect to get 0.5-1x106 cells). 

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24. Adjust cell number and proceed to FACS staining.