TITLE: Astrocyte isolation and culturing
AUTHORS: Julia F Riley

[DESCRIPTION]
This protocol details steps for preparing an astrocyte monoculture from mouse pups. From dissection to plated samples ready to be transfected, treated, lysed, or imaged, the protocol takes 20-25 days. 

[BEFORE_START]
The night before (could also do morning of, but makes your life easier):
Locate tools and put next to the microscope.
Aliquot Dissection Media (DM). Need two tubes- one 50mL-tube containing media needed to fill plates for the actual dissection (~30 mL) and another 50mL tube containing either 4.5 mL - 9 mL media (depending on how many pups are being dissected- 4.5 is sufficient for less than 6 pups) to put dissected cortices into for trypsin.
Breakdown of how the dissection media is used:
Tube 1 (~30 mL):
    o10 mL for each of two 10cm dishes to do the dissection in (20 mL total)
    o4 mL for each of two dishes to rinse pup heads after an EtOH wash, before dissection (8 mL total)
Tube 2, for dissected corticies:
    oFor less than 6 pups, 4.5 mL (and add 500 µL 2.5 % (v/v) trypsin for digestion)
    oFor more than 6 pups, 9 mL (and add 1 mL 2.5 % (v/v) trypsin for digestion)
Aliquot glial media:
4 mL per flask.
15 mL for the three washes following trypsin digestion.
5 mL to neutralize trypsin for spin.
Poly-D-Lysine coat flasks (1 flask/pup you plan to dissect) and store at 4 °C  :
Mix Poly-D-Lysine (ThermoFisher, ) with PBS -Ca2+, -Mg2+
You can also do this the morning of the dissection, but they must sit for 60 min at room temperature to allow for sufficient coating.

The morning of the dissection:

Submerge tools in EtOH.
Make 4 dissection media filled plates and 1 EtOH plate:
One 5cm dish (100 % (v/v) EtOH)
Two 5cm dishes (4 mL DM each)
Two 10cm dishes (10 mL DM each)
3. Place 50mL tubes containing DM on ice.














[STEPS]
SECTION: Dissection
1. Sacrifice mouse pups postnatal day (P) 1-3 via isoflourane overdose. 
SECTION: Dissection
2. Decapitate pups and transfer heads to a dish with 100 % (v/v) EtOH. Swirl to clean. 

SECTION: Dissection
4. Transfer heads to a new 10cm dish and place whole dish in a cell culture dish with a wider diameter. The surrounding dish should then be filled with ice to keep the heads chilled while dissecting.

SECTION: Dissection
5. Using the dissection microscope to magnify the procedure, remove skin from heads by tearing with forceps and pushing it down and to the side
SECTION: Dissection
6. Using spring-loaded scissors, make a sagittal cut through the skull, down the median plane, starting from the base near where the spinal cord would be. At the end of the cut, twist the scissors slightly so they’re diagonal behind the eye sockets and make one more, very short cut on either side. This helps you open up the skull.
SECTION: Dissection
7. Make two coronal cuts in the middle of the skull where the skull plates meet on either side of your original sagittal cut.
SECTION: Dissection
8. Using forceps, Gently open up the skull flaps you just created. If you’re taking cortex with you, you can gently slide the forceps under the skull and move it back and forth to disconnect them.
SECTION: Dissection
9. Once the ventral half of the skull is open, push off the dorsal part a little bit.
SECTION: Dissection
10. Dig your forceps diagonally under the brain and into the underneath part of the skull, toward each other. Once they’re under, firmly pull apart and the brain will come free.
SECTION: Dissection
11. Move the brains to a fresh dish on ice once they’re free of the skulls.
SECTION: Dissection
12. Bisect the brains so you have the two hemispheres. Unroll them to open them up one at a time, with the inside of the hemisphere initially facing up. Can make a cut with the forceps where the crease is that makes them re-roll to prevent this.
SECTION: Dissection
13. Remove all of the meninges from the inside. Also remove the hippocampus.
SECTION: Dissection
14. Flip hemisphere. Remove the rest of the meninges. Remove cortex area of the hemisphere, being careful to leave midbrain components.
SECTION: Dissection
15. 10.  Put cortices into the tube of dissection media. 
SECTION: Plating brain cells in flasks
16. Using a pasteur pipette and bulb, triturate cortices in the dissection media a few times to make the chunks of cortex a little smaller.
SECTION: Plating brain cells in flasks
17. Add 0.5 mL 2.5 % (v/v) trypsin per 4.5 mL dissection media and incubate for 7 min .

SECTION: Plating brain cells in flasks
18. Add 5 mL glial maintenance media to neutralize trypsin and centrifuge for 4 min at 1.1gxs.

SECTION: Plating brain cells in flasks
19. While cells are spinning, remove PDL solution from pre-prepared flasks (see Before Start section of protocol) and rinse 2x with sterile ddH2O. Put 3 mL media in each flask.

SECTION: Plating brain cells in flasks
20. GENTLY suck off the trypsin with a serological pipette and replace with 5 mL DMEM to wash cortices. Wait for the cortices to sink back to the bottom. 

SECTION: Plating brain cells in flasks
22. After the final wash, add 2 mL glial media.

SECTION: Plating brain cells in flasks
23. Homogenize with a Pasteur pipette attached to a bulb, and then using a P1000.
SECTION: Plating brain cells in flasks
24. Put cell suspension through   cell strainer.
 


SECTION: Plating brain cells in flasks
25. Supplement to desired volume with more media and mix by gently inverting. 
SECTION: Plating brain cells in flasks
26. 10.  Plate cells on T25 flasks.
SECTION: Glia maintenance
27. ~6-10 hours after dissecting, firmly whack the side of each flask with your palm one time to loosen cell debris. Immediately following this, replace media.
SECTION: Glia maintenance
28. Change media every 4320 min .
 

 



SECTION: Purifying astrocytes
29. Depending on the type of T25, either tighten cap so it’s airtight OR parafilm over the top of the cap and around the sides to ensure no air exchange and maintain 5% CO2 within the flask.
SECTION: Purifying astrocytes
30. Tape flask to a shaker, ensuring media is covering cells.  
SECTION: Purifying astrocytes
31. Shake for 240 min - 360 min at 37 °C, 250 rpm.

SECTION: Plating cultured glia
32. Prepare new PDL surfaces for glia at least 60 min before starting.
 


SECTION: Plating cultured glia
33. Warm 2.5 mL 0.05 % (v/v) Trypsin-EDTA and glial maintenance media in T25 flasks.  
 



SECTION: Plating cultured glia
34. Remove flasks from shaker and hit them firmly with your palm ~3-4 times to help dislodge stubborn contaminating cell types.
SECTION: Plating cultured glia
35. Aspirate media from flasks. 
SECTION: Plating cultured glia
37. Add 1.5mL trypsin-EDTA to each flask and put in the incubator for 7 min - 8 min. 

SECTION: Plating cultured glia
38. Remove flask- when cells are ready to come off, they should look shriveled but still attached under a microscope.
SECTION: Plating cultured glia
39. Smack the flask firmly with your palm 4-6 times to knock cells loose.
SECTION: Plating cultured glia
42. Add glial maintenance media ~1:1 to the trypsin-EDTA cell suspension in the 50mL conical to neutralize trypsin. 
SECTION: Plating cultured glia
44. Aspirate trypsin and resuspend cells in glial media using a p1000. 
 

SECTION: Plating cultured glia
45. Plate astrocytes at 175,000 LIVE cells per imaging plate.
SECTION: Dissection
3. Transfer heads to dish of dissection media and swirl again. Do this one more time.
SECTION: Plating brain cells in flasks
21. Repeat DMEM wash two more times for a total of three washes.
SECTION: Plating cultured glia
36. Wash 2x with PBS w/o magnesium and calcium and aspirate.
SECTION: Plating cultured glia
40. Use a 5mL strippette to get in the flask and rinse the rest of the cells off 
SECTION: Plating cultured glia
41. Remove cell suspension and put in a 50mL conical.
SECTION: Plating cultured glia
43. Spin at 1.1 gxs for 4 min.