Mouse Whole Brain Processing for Microglia Isolation, Cell Separation, and Flow Cytometry

 

Reagent and Instrument List

1X Trypsin 0.25% Complete RPMI (RPMI-1640, with 10% Fetal Bovine Serum, 1% Pen-Strep) 70 μm cell strainer

10X PBS  Percoll (Optional for removing myelinated cells) Various collection tubes/conical tubes

Protocol

Note: Use aseptic techniques if you need to sort and grow cells in culture.

 

Harvesting the brain

1. If the mouse is >2 weeks old, perfuse the mouse with 10 mL 1X PBS through the left ventricle prior to brain harvest.
2. Beginning from the brain stem, cut upward along the sagittal suture as to not damage the brain.
   Note: Sagittal Suture is a connective joint that splits the two halves of the skull.
3. Peel the two halves of the skull away to the side.
4. Using tweezers, scoop out the brain and place into a petri dish with cold PBS.

Enzymatic digestion

Note: Due to the effect of trypsin digestion on certain surface antigen epitopes, you may use a dounce homogenizer in lieu of trypsinization in step 7. See Garcia JA et al. below for additional information on this procedure.

 

5. Gently separate the brain into 6-8 pieces.
6. Transfer the segments of the brain into a 50 mL polypropylene conical tube.
7. Add 10 mL of trypsin to the tube containing the brain and incubate at 37°C in an incubator or water bath. Every 5 minutes, gently invert the tube or gently pipette the tissue.
   Note: Do NOT leave in trypsin for more than 15 minutes.
8. After 10-15 minutes, triturate any clumps until mostly dissociated and add 20-30 mL of complete RPMI. To triturate, you may pipet up and down using either 12 or 50 mL serological pipettes.
9. Filter the suspension using a 70 μm cell strainer over a clean tube 3 times. Then, gently homogenize any remaining large clumps using a pipette.
10. Spin down cells at 300 x g for 5 minutes at room temperature. Gently discard the supernatant.
11. Pick option A or B below based on your desire to remove myelin from the homogenate.
    1. If you do not wish to remove myelin: Resuspend cells in PBS or desired buffer/media. Proceed to downstream application or analysis such as cell separation, fluorescence activated cell sorting (FACS), or flow cytometry.
    2. If you wish to remove myelin: Prepare the various Percoll gradient solutions and resuspend the cell pellet in 4 mL/brain of 37% Percoll at room temperature. See the next section for steps on myelin removal. Place on ice until you are ready to proceed.

Optional steps: Myelin Removal for Microglia, Leukocyte Isolation

Note: If processing more than 1 brain, do NOT scale up in the same tube. Use multiple 15 mL polypropylene conical tubes to process multiple brains (1 brain per tube). 

 

Prepare several Percoll gradient solutions (warmed to room temperature):

• Stock Isotonic Percoll (SIP): Mix 1 part 10X PBS and 9 parts percoll.
• 70% Percoll: 7 mL SIP + 2.0 mL 1X PBS
• 37% Percoll: 3.7 mL SIP + 5.3 mL 1X PBS
• 30% Percoll: 3.0 mL SIP +6.0 mL 1X PBS

12. Transfer 4 mL 70% Percoll into a 15 mL polypropylene conical tube.
13. Slowly layer the 4 mL of homogenized cell suspension (in 37% Percoll from step 11b) into the tube containing 4 mL 70% Percoll (from step 12). The density gradient will cause the 37% Percoll solution to layer on top of the 70% Percoll.
14. Gently layer 4 mL of 30% Percoll into the tube followed by 2 mL of 1X PBS to complete the density gradient. The final gradient should layer sequentially from top to bottom: 
    • 2 mL 1X PBS
    • 4 mL of 30% Percoll
    • 4 mL of cells in 37% Percoll
    • 4 mL of 70% Percoll
15. Centrifuge gradient for 40 minutes at 300 x g at room temperature WITHOUT brakes to avoid agitating the density gradient and the separation between layers.
16. With a transfer pipette, remove the interphase between 1X PBS and 30% Percoll. This layer contains the myelin.
17. Using another transfer pipette, remove the interphase between 70% and 37% Percoll. This layer contains microglia and leukocytes. Do NOT discard! Transfer these cells to a fresh 15 mL conical tube.
18. Add 3X the volume of 1X PBS to the microglia cells to dilute the Percoll and centrifuge for 5 minutes at 365 x g at room temperature.
19. Resuspend the cell pellet in desired volume of 1X PBS and proceed to cell counting if desired.
20. Proceed to downstream application such as cell separation, fluorescence activated cell sorting (FACS), or flow cytometry analysis. 

Representative Data

 

 

Representative data using positive selection of P2RY12+ cells using the MojoSort™ Mouse P2RY12 Selection Kit on adult C57BL/6 mouse brain prepared using brain processing, Percoll gradient isolation procedure above. Cells were stained with anti-mouse P2RY12 (clone S16007D) PE and anti-mouse CD11b (clone M1/70) Brilliant Violet 421™ (left) or anti-mouse CX3CR1 (clone SA011F11) APC (right). Dead cells were excluded by 7-AAD.

Additional references: Garcia JA. et al. 2014. Curr Protoc Immunol.