Jun 11, 2026
Primary Astrocyte Culture from Postnatal Day 1 (P1) Pups
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Protocol Citation: Yue Ma 2026. Primary Astrocyte Culture from Postnatal Day 1 (P1) Pups. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnpxqyv5d/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: June 11, 2026
Last Modified: June 15, 2026
Protocol Integer ID: 318999
Keywords: ASAPCRN, primary astrocyte culture from postnatal day, primary astrocyte culture, microglia, oligodendrocyte, postnatal day, pups newborn littermate, cortex
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000592
Abstract
Newborn littermates were dissected at postnatal day 1 (P1), cortex were digested, triturated, and plated in the flasks, the microglia and oligodendrocytes were removed at 7 days in vitro (DIV) by shaking.
Materials
- Autoclave dissection tools**
- Coat tissue culture flask:**
- 50 ug/mL Poly D lysine (PDL)
- Astrocyte medium:**
- DMEM/F12
- 10% heat-inactivated fetal bovine serum
- 1% Penicillin/Streptomycin
- Dissociation medium (500 ml):**
- 2.6 g K2SO4 (30 mM final)
- 6.4 g Na2SO4 (90 mM final)
- 2.9 ml MgCl2 1M (5.8 mM final)
- 125 ul CaCl2 1M (0.25 mM final)
- 800 ul HEPES 1M (1.6 mM final)
- 1 ml NaOH 0.1M (0.2 mM final)
- 0.5 ml Phenol Red
- Digestion medium (15 ml):**
- 15 mg L-Cysteine (Sigma C7477)
- 14.1 ml dissociation media
- 180 ul 30% glucose
- 150 Units papain
- Protease inhibitor solution (15 ml):**
- 14.7 ml dissociation media
- 180 ul 30% glucose
- 150 ul trypsin inhibitor
- Trituration medium (30 ml):**
- 26.4 ml MEM
- 360 ul 30% glucose
- 300 ul GlutaMAX (L-glutamine 200 mM)
- 3 ml heat inactivated horse serum
Before start
- Pre-dissection prepares**
Pre-dissection prepares
Autoclave dissection tools
Coat tissue culture flask: Cover the flask surface with 50 µg/mL Poly D lysine (PDL), incubate at 37 °C for 1 hour in tissue culture incubator, rinse with sterile H₂O and let dry in tissue culture hood, store in tissue culture incubator until plating cells.
Dissociation medium (500 ml):
2.6 g K₂SO₄ (30 mM final)
6.4 g Na₂SO₄ (90 mM final)
2.9 ml MgCl₂ 1M (5.8 mM final)
125 ul CaCl₂ 1M (0.25 mM final)
800 ul HEPES 1M (1.6 mM final)
1 ml NaOH 0.1M (0.2 mM final)
0.5 ml Phenol Red
Adjust volume to 500 ml with H₂O, check pH ~7.4, filter with 0.22 µm filter, store at 4°C, good for 4 months.
Digestion medium (15 ml):
15 mg L-Cysteine (Sigma C7477)
14.1 ml dissociation media
180 ul 30% glucose
150 Units papain (volume depends on batch)
Adjust pH to 7.4 with 5M NaOH, heat for 30 minutes in 37°C water bath to activate enzyme, filter with 0.22 µm filter.
Protease inhibitor solution (15 ml):
14.7 ml dissociation media
180 ul 30% glucose
150 ul trypsin inhibitor
Filter with 0.22 µm filter.
Trituration medium (30 ml):
26.4 ml MEM
360 ul 30% glucose
300 ul GlutaMAX (L-glutamine 200 mM)
3 ml heat inactivated horse serum
Filter with 0.22 µm filter.
Brain dissection
Carefully open skull, remove brain and place in drop of pre-warmed HBSS. Remove meninges then cut out a piece of cortex. Place pieces of cortex in appropriate culture dish with pre-warmed HBSS.
Place the tissue in 2 ml of digestion media. Use a scalpel to chop tissue chunks into small pieces.
Transfer the tissue using a wide-bore glass pipette to a 15ml tube. Gently agitate before incubating horizontally in bead bath set at 34°C for 30 minutes. After 15 minutes, gently agitate again.
Centrifuge for 2 minutes at 1500rpm. Keep the pellet.
Resuspend the pellet in 2 ml Protease Inhibitor. Let the suspension stand horizontally for 10 min at RT.
Centrifuge the suspension for 2 minutes at 1500rpm. Remove the supernatant containing Protease Inhibitor.
Resuspend the pellet in 4 ml of trituration medium. Start the trituration by pipetting up and down (10x-20x) with 2 ml glass pipette.
Allow tissue to settle down, remove supernatant (with the cells) and transfer to 50 ml conical tube. Repeat trituration until few large pieces remain (around 4 to 5 times).
Centrifuge the tube containing triturated supernatant for 3 min at 1500rpm. Remove supernatant, keep the pellet with the cells.
Resuspend the pellet in 5ml of Astrocyte medium.
Count the cells.
Plate 3-5 x 10^6^ cells per T25 flask in 6 mL Astrocyte Medium.
Incubate at 37°C in TC hood, change the medium next day after plating and every other day.
First Cell Split (removing microglia and OPCs)
Remove microglia from astrocyte culture by shaking at 180 rpm for 30 min on an orbital shaker.
Remove and discard media, replacing with 6mL fresh pre-warmed astrocyte medium, and shake for 6 hours at 240rpm to remove oligodendrocyte precursor cells (OPCs) from the culture.
Rinse astrocytes twice with fresh medium and then add trypsin and incubate at 37°C in the TC incubator for 5 minutes.
Add 5mL of medium to flasks and transfer to 50mL falcon tube.
Spin cells down at 180 x g for 5 min.
Aspirate off supernatant and add 20 mL of fresh pre-warmed astrocyte medium.
Add 20 mL medium / astrocyte mixture to T75 flask and incubate at 37°C in the TC incubator.
Change medium every 2-3 days.
Split cells 1:2 every ~7 days
Notes: Astrocytes should be used for experiments at DIV 21 to 28. Astrocytes should not be split more than 3 times.