TITLE: Microglia isolation from mouse and culture (modified from UCSD)
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details microglia isolation from mouse and culture (modified from UCSD).

[GUIDELINES]

General notes

(Maximum 6 mice per preparation; protocol described below for 6 brains; ~300,000 cells per 7/week old).
For high yield better use really young mice, let’s say with P7-14 you may get a lot more than above!
Mechanical dissociation >> enzymatic dissociation.
Keep everything at 4 °C.
In vitro: 150k cells/ well (one brain/well) in a 48 well plate).
Will get much more microglia with pups à use microglia from pups to test antibodies and for general practice. 


[STEPS]
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
1. Deeply anaesthetize with CO2.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
1.1. Can also use euthanasia.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
2. Perfuse intracardially with ice-cold PBS (~10 to 20ml, liver gets white).
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
3. Remove whole brain and place into staining buffer (HBSS 1X – Life Technologies, 14175-095; 1% BSA, 1 millimolar (mM) EDTA) on ice.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
3.1. Place in 15 ml falcon tube.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
4. Dissect brain into 6 pieces.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
4.1. Place brain into mini cell culture dish with HBSS buffer.
 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
4.2. Remove spinal cord completely.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
4.3. Cut with dissecting blade.

 Remove cerebellum
 Cut forebrain into several pieces
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
4.4. Put brain pieces into 15ml falcon with HBSS.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
5. Gently homogenize in staining buffer (~10 mL) using 2 mL polytetrafluoroethylene pestle (Wheaton, 358026), for six brains (8-week-old) polypropylene round-bottom tube (Corning, 352059).

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
5.1. Put brain into clear round bottom test tube (15 ml).
 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
5.2. Gently go up and down with pestle a don’t press down on bottom of tube.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
5.3. Keep tubes on ice.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
5.4. Homogenize for roughly 30 min.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
6. When supernatant is cloudy, spin briefly to 48 g, store supernatant in collection tube.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
7. Then continue to homogenize the brain chunks in 15 ml tubes (Corning, 352063).

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
6.1. Acceleration=5 
 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
6.2. Deceleration=5
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
6.3. Wait until centrifuge gets to 48 and then immediately stop the spinning.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
6.4. Put supernatant into a separate collection tube (label tube); 15 ml falcon tube.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
6.5. Add HBSS + EDTA+BSA buffer into tube with brain chunks.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
7.1. Homogenize for roughly 20 min.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
8. Spin briefly to 48 g when supernatant is cloudy roughly 30 min each time.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
8.1. Transfer supernatant to collection tube.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
8.2. Put brain chunks into a smaller tube (corning 352063).
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
9. Add buffer to brain chunks and repeat cycle until procedure becomes inefficient (You should have whitish stuff and supernatant is clear) Should have between 15 and 25 ml whitish to yellowish supernatant per brain.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
9.1. Supernatant should become clear towards the end of dissociation (although there will still be white chunks of microglia left).
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
10. Lastly, transfer supernatant in 2 ml glass mortar (Wheaton, 358004), do once up & down with cell suspension SLOWLY.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
10.1. Step is to remove and separate chunks of microglia.
 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
10.2. Add 1.6-1.8 of supernatant into glass mortar.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
10.3. Use pestle to push down gently into liquid and then remove pestle and transfer supernatant to a new collection tube (50 ml falcon tube). 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
10.4. Keep adding 2 mL of the supernatant from the collection tube to the glass mortar and repeat this process for remaining supernatant.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
11. Filter homogenate onto 70 µm cell strainer (BD Falcon 352350).
 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
11.1. Fill homogenate to 40 mL with HBSS + EDTA +BSA in the 50 ml falcon tube.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
11.2. Can switch to a new filter if it gets clogged. 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
12. Centrifuge for 12 min at 400 x g at 18 °C (step 5 acceleration, step 3 brakes).



SECTION: Microglia isolation from mouse and culture (modified from UCSD)
12.1. Pour out supernatant.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
13. Prepare Percoll (Sigma, P4937).


SECTION: Microglia isolation from mouse and culture (modified from UCSD)
13.1. 45 mL percoll + 5 mL 10x HBSS (for 6 brains).

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
14. Resuspend pelleted homogenate in 6 mL (per brain) of 37% isotonic Percoll in 15 ml centrifuge tube.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
14.1. 37% percoll

13.3 percoll (from step 13)
 22.7 1x HBSS
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
14.2. 70% percoll

23.1 percoll (from step 13)
9.9 1x HBSS 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
15. Underlay with 5 mL of 70% isotonic Percoll.
 

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
15.1. Use a 5 mL pipette for underlay.


SECTION: Microglia isolation from mouse and culture (modified from UCSD)
15.2. Move pipette up very slow as you add more volume but make sure you are not too close to interphase.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
16. Centrifuge at 600 x g for 40 min at 16 °C-18 °C, with “0“ acceleration and deceleration (!).

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
17. Remove myelin and everything to about 3 mL above the interphase.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
18. Recover cells at the 37%–70% Percoll interphase, this should be ~2.5-3 ml.
 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
17.1. Remove 1-2 ml of debris on top.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
18.1. First put 2 mL of HBSS+EDTA+BSA into a collection tube (15 ml falcon tube).

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
18.2. Then get interphase (circle around with pipette).
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
18.3. Then get 2-3 ml above the interphase (don't collect liquid below interphase!). 
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
18.4. Remove 2 mL of this separated cells into another tube for unstained control cells for FACS.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
19. Centrifuge for 10 min at 400 x g in HBSS 1X/MG media (1:1), remove supernatant.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
19.1. Fill tube with HBSS before spinning.
SECTION: Microglia isolation from mouse and culture (modified from UCSD)
20. Repeat in 14 mL Falcon tube in HBSS once or twice.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
21. Resuspend in 300 µL of HBSS +EDTA + BSA.

SECTION: Microglia isolation from mouse and culture (modified from UCSD)
22. Store cells in fridge for staining.