Microglia isolation from mouse and culture (modified from UCSD)

Deeply anaesthetize with CO_2.

Can also use euthanasia.

Perfuse intracardially with ice-cold PBS (~10 to 20ml, liver gets white).

Remove whole brain and place into staining buffer (HBSS 1X – Life Technologies, 14175-095; 1% BSA, 1millimolar (mM) EDTA) On ice.

Place in 15 ml falcon tube.

Dissect brain into 6 pieces.

Place brain into mini cell culture dish with HBSS buffer.

Remove spinal cord completely.

Cut with dissecting blade.

• Remove cerebellum
• Cut forebrain into several pieces

Put brain pieces into 15ml falcon with HBSS.

Gently homogenize in staining buffer (~10mL) using 2mL polytetrafluoroethylene pestle (Wheaton, 358026), for six brains (8-week-old) polypropylene round-bottom tube (Corning, 352059).

Put brain into clear round bottom test tube (15 ml).

Gently go up and down with pestle a don’t press down on bottom of tube.

Keep tubes On ice.

Homogenize for roughly 0h 30m 0s.

When supernatant is cloudy, spin briefly to 48g, store supernatant in collection tube.

Acceleration=5

Deceleration=5

Wait until centrifuge gets to 48 and then immediately stop the spinning.

Put supernatant into a separate collection tube (label tube); 15 ml falcon tube.

Add HBSS + EDTA+BSA buffer into tube with brain chunks.

Then continue to homogenize the brain chunks in 15 ml tubes (Corning, 352063).

Homogenize for roughly 0h 20m 0s.

Spin briefly to 48g when supernatant is cloudy roughly 0h 30m 0s each time.

Transfer supernatant to collection tube.

Put brain chunks into a smaller tube (corning 352063).

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.

Supernatant should become clear towards the end of dissociation (although there will still be white chunks of microglia left).

Lastly, transfer supernatant in 2 ml glass mortar (Wheaton, 358004), do once up & down with cell suspension SLOWLY.

Step is to remove and separate chunks of microglia.

Add 1.6-1.8 of supernatant into glass mortar.

Use pestle to push down gently into liquid and then remove pestle and transfer supernatant to a new collection tube (50 ml falcon tube).

Keep adding 2mL of the supernatant from the collection tube to the glass mortar and repeat this process for remaining supernatant.

Filter homogenate onto 70 µm cell strainer (BD Falcon 352350).

Fill homogenate to 40mL with HBSS + EDTA +BSA in the 50 ml falcon tube.

Can switch to a new filter if it gets clogged.

Centrifuge for 0h 12m 0s at 400x g,0h 0m 0s at 18°C (step 5 acceleration, step 3 brakes).

Pour out supernatant.

Prepare Percoll (Sigma, P4937).

45mL percoll + 5mL 10x HBSS (for 6 brains).

Resuspend pelleted homogenate in 6mL (per brain) of 37% isotonic Percoll in 15 ml centrifuge tube.

37% percoll

• 13.3 percoll (from step 13)
• 22.7 1x HBSS

70% percoll

• 23.1 percoll (from step 13)
• 9.9 1x HBSS

Underlay with 5mL of 70% isotonic Percoll.

Use a 5 mL pipette for underlay.

Move pipette up very slow as you add more volume but make sure you are not too close to interphase.

Centrifuge at 600x g,0h 0m 0s for 0h 40m 0s at 16°C-18°C, with “0“ acceleration and deceleration (!).

Remove myelin and everything to about 3mL above the interphase.

Remove 1-2 ml of debris on top.

Recover cells at the 37%–70% Percoll interphase, this should be ~2.5-3 ml.

First put 2mL of HBSS+EDTA+BSA into a collection tube (15 ml falcon tube).

Then get interphase (circle around with pipette).

Then get 2-3 ml above the interphase (don't collect liquid below interphase!).

Remove 2mL of this separated cells into another tube for unstained control cells for FACS.

Centrifuge for 0h 10m 0s at 400x g,0h 0m 0s in HBSS 1X/MG media (1:1), remove supernatant.

Fill tube with HBSS before spinning.

Repeat in 14mL Falcon tube in HBSS once or twice.

Resuspend in 300µL of HBSS +EDTA + BSA.

Store cells in fridge for staining.