Jul 31, 2023

Public workspaceAstrocyte isolation and culturing

DOI
dx.doi.org/10.17504/protocols.io.e6nvwdp9wlmk/v1
  • 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Julia F Riley
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwdp9wlmk/v1
Protocol Citation: Julia F Riley 2023. Astrocyte isolation and culturing. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdp9wlmk/v1
Manuscript citation:
Schildge S, Bohrer C, Beck K, Schachtrup C. Isolation and culture of mouse cortical astrocytes. J Vis Exp. 2013 Jan 19;(71):50079. doi: 10.3791/50079. PMID: 23380713; PMCID: PMC3582677.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83014
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
Abstract
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):
  1. Locate tools and put next to the microscope.
  2. Aliquot Dissection Media (DM). Need two tubes- one 50mL-tube containing media needed to fill plates for the actual dissection (~Amount30 mL ) and another 50mL tube containing either Amount4.5 mL - Amount9 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 (~Amount30 mL ):
oAmount10 mL for each of two 10cm dishes to do the dissection in (Amount20 mL total)
oAmount4 mL for each of two dishes to rinse pup heads after an EtOH wash, before dissection (Amount8 mL total)
  • Tube 2, for dissected corticies:
oFor less than 6 pups, Amount4.5 mL (and add Amount500 µL Concentration2.5 % (v/v) trypsin for digestion)
oFor more than 6 pups, Amount9 mL (and add Amount1 mL Concentration2.5 % (v/v) trypsin for digestion)
  1. Aliquot glial media:
  • Amount4 mL per flask.
  • Amount15 mL for the three washes following trypsin digestion.
  • Amount5 mL to neutralize trypsin for spin.
  1. Poly-D-Lysine coat flasks (1 flask/pup you plan to dissect) and store at Temperature4 °C DurationOvernight :
  • Mix Poly-D-Lysine (ThermoFisher, ) with PBS -Ca2+, -Mg2+
  • You can also do this the morning of the dissection, but they must sit for Duration01:00:00 at room temperature to allow for sufficient coating.

The morning of the dissection:

  1. Submerge tools in EtOH.
  2. Make 4 dissection media filled plates and 1 EtOH plate:
  • One 5cm dish (Concentration100 % (v/v) Sterile EtOH)
  • Two 5cm dishes (Amount4 mL DM each)
  • Two 10cm dishes (Amount10 mL DM each)
3. Place 50mL tubes containing DM on ice.













Dissection
Dissection
Sacrifice mouse pups postnatal day (P) 1-3 via isoflourane overdose.
Decapitate pups and transfer heads to a dish with Concentration100 % (v/v) Sterile EtOH. Swirl to clean.

Transfer heads to dish of dissection media and swirl again. Do this one more time.
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.

Using the dissection microscope to magnify the procedure, remove skin from heads by tearing with forceps and pushing it down and to the side
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.
Make two coronal cuts in the middle of the skull where the skull plates meet on either side of your original sagittal cut.
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.
Once the ventral half of the skull is open, push off the dorsal part a little bit.
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.
Move the brains to a fresh dish on ice once they’re free of the skulls.
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.
Remove all of the meninges from the inside. Also remove the hippocampus.
Flip hemisphere. Remove the rest of the meninges. Remove cortex area of the hemisphere, being careful to leave midbrain components.
10. Put cortices into the tube of dissection media.
Plating brain cells in flasks
Plating brain cells in flasks
Using a pasteur pipette and bulb, triturate cortices in the dissection media a few times to make the chunks of cortex a little smaller.
Add Amount0.5 mL Concentration2.5 % (v/v) trypsin per Amount4.5 mL dissection media and incubate for Duration00:07:00 .

7m
Add Amount5 mL glial maintenance media to neutralize trypsin and centrifuge for Duration00:04:00 at 1.1gxs.

4m
While cells are spinning, remove PDL solution from pre-prepared flasks (see Before Start section of protocol) and rinse 2x with sterile ddH2O. Put Amount3 mL media in each flask.

GENTLY suck off the trypsin with a serological pipette and replace with Amount5 mL DMEM to wash cortices. Wait for the cortices to sink back to the bottom.

Repeat DMEM wash two more times for a total of three washes.
After the final wash, add Amount2 mL glial media.

Homogenize with a Pasteur pipette attached to a bulb, and then using a P1000.
Put cell suspension through Thikness100 µm cell strainer.
Note
any cell strainer with mesh size greater than Thikness100 µm is sufficient



Supplement to desired volume with more media and mix by gently inverting.
10. Plate cells on T25 flasks.
Glia maintenance
Glia maintenance
1w 6d
1w 6d
~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.
Change media every Duration72:00:00 .
Note
Could be more or less depending on what cells look like they need, but the cell strainer and same-day whack and media change helps eliminate initial debris.

Note
Cells should look good around 10-14 days.



3d
Purifying astrocytes
Purifying astrocytes
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.
Tape flask to a shaker, ensuring media is covering cells.
Shake for Duration04:00:00 - Duration06:00:00 at Temperature37 °C , 250 rpm.

6h
Plating cultured glia
Plating cultured glia
1h 12m
1h 12m
Prepare new PDL surfaces for glia at least Duration01:00:00 before starting.
Note
can also do this step the night before and put plates in refrigerator


1h
Warm Amount2.5 mL Concentration0.05 % (v/v) Trypsin-EDTA and glial maintenance media in T25 flasks.
Note
Make sure it’s super warm before you use it! This helps the cells come off easier.



Remove flasks from shaker and hit them firmly with your palm ~3-4 times to help dislodge stubborn contaminating cell types.
Aspirate media from flasks.
Wash 2x with PBS w/o magnesium and calcium and aspirate.
Add 1.5mL trypsin-EDTA to each flask and put in the incubator for Duration00:07:00 - Duration00:08:00 .

8m
Remove flask- when cells are ready to come off, they should look shriveled but still attached under a microscope.
Smack the flask firmly with your palm 4-6 times to knock cells loose.
Use a 5mL strippette to get in the flask and rinse the rest of the cells off
Remove cell suspension and put in a 50mL conical.
Add glial maintenance media ~1:1 to the trypsin-EDTA cell suspension in the 50mL conical to neutralize trypsin.
Spin at 1.1 gxs for Duration00:04:00 .

4m
Aspirate trypsin and resuspend cells in glial media using a p1000.
Note
To do this, add Amount1 mL of media and resuspend with a p1000 until homogenous, then add more glial media (~Amount4 mL more) to bring cell concentration into a range where cell count accuracy is attainable by the countess cell counter. Invert to mix and count cells. Remember to record % viability.


Plate astrocytes at 175,000 LIVE cells per imaging plate.