TITLE: PRIMARY GLIA ISOLATION AND CULTURE PROTOCOL
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the isolation of primary glia from cortex.

[STEPS]
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1. Before dissection:
SECTION: Primary Glia Isolation From Cortex Protocol
1.1. Clean and autoclave all dissection tools (scissors, forceps, spatulas, razor blades) prior to use.

SECTION: Primary Glia Isolation From Cortex Protocol
1.2. Prepare dishes or plates.

Minimum of 60 min in 37 °C incubator.

SECTION: Primary Glia Isolation From Cortex Protocol
1.3. Have solutions warmed, equilibrating, and prepared prior to starting dissection (plating media, digestion solution, digestion inhibition solution).

Sterile filter digestion and inhibition solutions prior to use.

SECTION: Primary Glia Isolation From Cortex Protocol
1.4. Flame polish autoclaved 9” Pasteur pipettes.

SECTION: Primary Glia Isolation From Cortex Protocol
2. Dissection:
SECTION: Primary Glia Isolation From Cortex Protocol
2.1. In laminar flow hood: have aluminum foil for mice, Kimwipes or paper towels for dissection, tools, ice bucket and Brain Bits Hibernate A (BB HA).

SECTION: Primary Glia Isolation From Cortex Protocol
2.2. Begin dissection (steps may be done simultaneously on 6-8 pups or sequentially on each pup).

Remove tools from alcohol
Decapitate pup/s with scissors
Use razor to make a mid-sagittal incision only penetrating the skin
Use razor to make a small mid-sagittal incision in the skull, then press down hard hemisecting the brain and skull. Push apart.
Dip blunt dissecting spatulas into the wash solution. Scoop out brain hemisphere, severing the olfactory bulb for ease
Separate and isolate cortex.
Place in chilled BB HA solution
Using fine forceps, remove meninges from cortical surface

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2.3. Keep on ice until ready to place cortex into warmed and sterile filtered digestion solution.

SECTION: Primary Glia Isolation From Cortex Protocol
3. Digestion:
SECTION: Primary Glia Isolation From Cortex Protocol
3.1. Using 10 mL serological pipette, transfer cortices from BB HA to digestion solution.

SECTION: Primary Glia Isolation From Cortex Protocol
3.2. Incubate in 37 °C water bath for 10 min-15 min, with intermediate mixing.

SECTION: Primary Glia Isolation From Cortex Protocol
3.3. During this time:

Ensure plates/dishes are ready
Prepare trypan blue Eppendorf tube (150 µLTB + 50 µL cells) to count

SECTION: Primary Glia Isolation From Cortex Protocol
4. Inhibition + Triturate:
SECTION: Primary Glia Isolation From Cortex Protocol
4.1. Following digestion incubation, gently remove cortices with 10 mL serological pipette and place into 15 mL conical tube.

SECTION: Primary Glia Isolation From Cortex Protocol
4.2. Wash cortices 3x with inhibition solution (3-4mL/wash).

SECTION: Primary Glia Isolation From Cortex Protocol
4.3. Then add final 4 mL-5 mLinhibition solution and triturate cortices gently using fire polished pasteur pipette.

SECTION: Primary Glia Isolation From Cortex Protocol
4.4. Once triturated, allow any undissociated tissue to sink to the bottom, gently transfer remaining suspension to fresh 15 mL tube.

SECTION: Primary Glia Isolation From Cortex Protocol
4.5. Pass remaining supernatant through 70 m and 40 m cell strainers to isolate single cell suspension.

SECTION: Primary Glia Isolation From Cortex Protocol
4.6. Pull 50 µL aliquot for counting, then centrifuge at 300 x g, 4 min, 4 °C.

SECTION: Primary Glia Isolation From Cortex Protocol
5. Count cells:
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5.1. Make up the 200 µL (1:4 dilution of cells) trypan blue mixture, load 10 µL to hemacytometer, and count 4 quadrants.

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5.2. Calculate desired concentration of cells/mL.

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6. Plate cells:
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6.1. Dilute cells with appropriate amount of pre-equilibrated plating media, (20S: DMEM, 1 mM Sodium pyruvate, Glutamax, Penicillin-streptomycin and FBS-20%) to get desired cell concentration.

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6.2. Plate 12,000,000 cells in each matrigel coated T75 flask.

SECTION: Primary Glia Isolation From Cortex Protocol
7. Microglia isolation and culture:
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7.1. To obtain primary microglia, shake confluent T75 flask at  .

SECTION: Primary Glia Isolation From Cortex Protocol
7.2. Centrifuge suspended microglia at 300 x g, 5 min and resuspended in 20S plating medium followed by filtering through 70 µm cell strainer. 

SECTION: Primary Glia Isolation From Cortex Protocol
7.3. Plate cells at desired concentration 2880 min prior to experiment.

SECTION: Primary Glia Isolation From Cortex Protocol
8. Oligodendrocytes isolation and culture:
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8.1. After microglia have been removed from T75 flasks, replace media and shake at  .  

SECTION: Primary Glia Isolation From Cortex Protocol
8.2. Filter suspended oligodendrocytes using 40 m cell strainer and centrifuge at 200 x g, 10 min.
 
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8.3. Resuspend cell in OPC media. 

 OPC media:
 
 
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8.4. Plate cells 7-10 days prior to experiment.

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9. Astrocytes isolation and culture:
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9.1. After microglia and oligodendrocytes have been removed from T75 flasks, wash the remaining attached cells (astrocytes) twice with PBS detach using 0.25% Trypsin-EDTA, add 5 ml NbAstro media and filter through 40 µm cell strainer and centrifuge at 300 x g, 10 min. 

SECTION: Primary Glia Isolation From Cortex Protocol
9.2. Resuspend pellet in NbAstro media and filter through 70 and then 40 µm cell strainer. 
 
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9.3. Plate astrocytes at 800,000 or 400,000 cells per well for 2-4 days before experiment for biochemical or immunocytochemical analysis, respectively.