May 05, 2026

Isolation and culture of primary mouse astrocytes and neurons

 Forked from Primary murine microglia, neurons and co-cultures
DOI
https://dx.doi.org/10.17504/protocols.io.3byl4pynrlo5/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4pynrlo5/v1
Protocol Citation: Sarah van Veen, Marta Montpeyó Garcia-Moreno 2026. Isolation and culture of primary mouse astrocytes and neurons . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4pynrlo5/v1
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: April 14, 2026
Last Modified: May 05, 2026
Protocol  Integer ID: 314989
Keywords: ASAPCRN, culture of primary mouse astrocyte, primary mouse astrocyte, primary murine astrocyte, neuron culture, neuron
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol details the methodology to establish primary murine astrocyte and neuron cultures.
Materials
Astrocyte growth media (AGM)
DMEM (GIBCO, Cat# 41965039)
10% FBS (PAN BioTech, Cat# P30-3306)
10 μM hydrocortisone (Sigma, Cat# H0888)
100 U mL−1 penicillin/streptomycin (Sigma, Cat# P4458)
5 μg mL−1 insulin (Sigma, Cat# I1882)
1 mM Na pyruvate (GIBCO, Cat# 11360-070)
5 μg mL−1 N-acetyl-L-cysteine (Sigma, Cat# A8199)

HBSS-HEPES buffer
490 mL HBSS 1× without CaCl₂ or MgCl₂ (GIBCO, Cat# 14175-053)
5 mL (10 mM final) HEPES, pH 7.3 (GIBCO, Cat# 15630-056)
5 mL (1% final) penicillin/streptomycin (GIBCO, Cat# 15140-122)

Enzyme solution (20 mL):
- 0.0064 g L-cysteine (Sigma)
- 20 mL DM
- 400 units papain (Worthington)
- Incubate 30’ at 37°C until solution is clear
- pH enzyme solution with 0.1N NaOH until it looks like DM again
- Syringe filter enzyme solution

Plating medium (50 mL):
- 430 mL MEM (1×) with Earle’s salts and L-glutamine (GIBCO, Cat# 31095029)
- 50 mL heat-inactivated horse serum (GIBCO, Cat# 26050088)
- 15 mL 20% glucose (Sigma, Cat# G7021; dissolved in Baxter water, filtered (0.22µm))
- 5 mL penicillin/streptomycin (GIBCO, Cat# 15140-122)

Neuronal Feeding Media (50 mL):
- 1 mL B27 (GIBCO, Cat# 17504044)
- 0.6 mL 1M glucose (Sigma, Cat# G7021)
- 0.1 mL 100x pen/strep (GIBCO, Cat# 15140-122)
- 0.125 mL 100x glutamax (Thermo Fisher, Cat# 35050038)
- 1 μL 1.25M ß-mercapto-ethanol (Sigma, Cat# M3148)
- 48.2 mL Neurobasal (GIBCO, Cat# 21103049) 

DNase (Sigma, Cat# DN25)

Poly-D Lysine (astrocytes: Sigma, Cat# P6407;neurons: GIBCO, Cat# A3890401)

Laminin (Sigma, Cat# L2020)

5-fluoro-2′-deoxyuridine (FUdR, 5 μM; Sigma, Cat# F0503)

Astrocyte culture
7h 50m
Coat 75 cm2 flasks
Add 10 mL of 20 µg/ml Poly-D-lysine (PDL) per flask.
30m
Incubate at 37 °C for 02:00:00 . Wash 3x with distilled H2O. Add AGM and allow to equilibrate in the incubator.

2h
Brain dissection, meninges removal and preparation of single cell suspension
3h
Sacrifice postnatal day 1 (P1) pups and wash the heads with sterile PBS
Remove meninges, olfactory bulb, cerebellum and brainstem.
Dissect the cortices in ice-cold astrocyte growth media (AGM), and carefully remove the meninges with fine foreceps
Use a flame-polished glass pipette (approximately half the original diameter) to homogenize the tissue by pipetting up and down 10-15 times
Further homogenize the tissue by passing through a 23G needle (10-15 times)
Once the tissue is homogenized, add 9 ml of AGM
Centrifuge at 300g for 10 min at room temperature
Discard the supernatant, add 1 ml AGM and repeat step 2.3 and 2.4.
Plate at a density of three brains per PDL-coated T75 flask
Same procedure for shake-off, transfection and co-culture used as in
Protocol
NAME

Astrocyte Isolation and Transfection for Astrocytes/Neuron co-culture assay

CREATED BY
Sarah van Veen

Neuron culture
3h 20m
Coat glass coverslips
2h
Washing glass cover slips
- Wash coverslips (cs) overnight in Nitric Acid (on wobbler, parafilm covered)
- Wash 3x 1 h in Baxter water
- Wash 1x in 70% Ethanol
- Keep cs in 70 % Ethanol ( parafilm covered)
- Place the washed cs in a 24-well plate using tweezers and let dry
PDL coating
- Use Poly-D Lysine (0.1 mg/ml)
- Add 300 µl/well/cs (24-well plate)
- Make sure the cs are not floating by pressing them slightly to the bottom with your pipet.
- Incubate overnight in the incubator (37°C)
Remove PDL coating and let dry the cs
- Remove the PDL
- Wash 3 times 1 h with Baxter water
- Let dry the 24-well plate with cs
- Keep at 4°C
Laminin coating
- Dilute the Laminin 1/1000 in Baxter water to a final concentration of 1 µg/ml
- Add 300 µl/well/cs  (24 well plate).
- Incubate in the incubator (at least 2h before seeding the primary neurons)
- Remove Laminin
- Wash once with plating medium (quick wash)
- Add 500 µl plating medium
- Put in incubator
Dissection and Trituration
Preheat plating medium at 37°C in water bath for at least 20 min.
Take a box of ice and place HBSS buffer on ice as well as a few small petri dishes with 1.5 ml HBBS-HEPES and one 15 ml tube filled with HBSS-HEPES.
Dissect cortices from P1 pups in ice-cold HBSS-HEPES buffer.
- Cut off heads of the pups and transfer them to a 10 cm petri dish with cold HBSS-HEPES
- Remove the brain from the skull
- Introduce a fine tweezer into the eyes to hold the head
- Remove the skin and the skull with a fine curved tweezer. Start at the olfactory bulbus and try the peel off in the direction of the hindbrain.
- Scoop the brain using a curved tweezer and transfer it to a small petri dish filled with 1.5 ml HBSS-HEPES and put it on ice.

Micro dissection of each brain by using the microscope (binocular) 
- Hold the hindbrain with tweezers
- Use sharp tweezers or small curved scissors to remove both hemispheres from the midbrain/ brainstem
- Gently remove meninges from the hemisphere by using tweezers
- Isolate the cortex and place in ice cold HBSS-HEPES
- Put the cortex in a small petri dish with HBSS-HEPES on ice and cut into smaller pieces and transfer the cortices to a 15ml falcon tube with ice cold HBSS-HEPES.
Carefully remove the excess of HBSS-HEPES  from the falcon tube containing the tissue until we are left with 9 ml, avoiding taking any tissue fragments (tissue will precipitate on the bottom of the tube)
Add 1 ml papain-based enzyme solution and 50 µl DNase (10 mg/ml)
Incubate at 37°C in the water bath for 20 min, mix periodically.
Note
Use this time to remove laminin and wash plates, and to prepare the Neurobasal (add the B27 and put in a flask inside the incubator to equilibrate temperature and CO2)

20m
Carefully remove enzyme solution from the tissue
Wash tissue 3x with 5 ml plating medium
Add 3 ml plating medium and dissociate the tissue with a narrow flamed Pasteur pipette
- Gently suck up all tissue, first a little medium, hold the bottom of the pipette close to the bottom of the 15 ml falcon tube (without touching)
- Pipette the tissue up and down for 5 to 10 times (don’t blow air bubbles into the medium with the tissue/cells) with ½ flame narrowed pipette, repeat with ¾ flame narrowed pipette.
- Centrifuge the dissociated CX single cells solutions @ RT for 5min @1000rpm 
- Carefully remove the medium from the cell pellet
- Add 1 ml of plating medium to the pellet and slowly resuspend the cells by pipetting up and down 3 times.
- Add 10 ml of plating medium
Count cells using a hemocytometer.
Plate 100,000/well onto glass coverslips, swirl the plate gently to spread the cells over the well,
incubate the plate for 30 min at RT.
1h
Place the cells in the incubator (37°C, 5% CO2) for 2 h
After 2 h replace the medium with Neuronal Feeding Media
48h after plating, treat cultures with 5-fluoro-2′-deoxyuridine (FUdR, 5 μM)
48h later, replace medium completely with fresh neuronal culture medium