Apr 15, 2025
Mouse Primary Astrocyte (mPA) isolation and culture
- 1Duke University
- Gersbach Lab
Protocol Citation: Samuel Reisman, Charles Gersbach 2025. Mouse Primary Astrocyte (mPA) isolation and culture . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbn7dqgpk/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 04, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126157
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocols describes methods for isolating and culturing primary astrocytes from mice.
Materials
Cytosine arabinoside (AraC) (1000X)
Dissolve 4.86 mg AraC (Sigma, C1768) in 2 ml distilled water to make 1,000x stock solution of 10 mM and filter the solution with 0.22 µm filters. Store at -20 °C and protect from light.
Hydrocortisone (1000X)
Stock concentration: 10mM in DMSO. Dissolve 0.108g of Hydrocortisone (sigma H-0888) in 30ml of DMSO. Filter with 0.22 µm filters and save at -20.
Lo Ovomucoid (10X)
To 150ml of DPBS (Thermo Fisher Scientific, 14287-080), add 3 g BSA (Sigma A8806). Mix well. Add 3 g Trypsin inhibitor (Worthington, LS003086) and mix to dissolve. Adjust pH to 7.4; requires the addition of approximately 1ml of 1N NaOH. When completely dissolved, bring to 200 ml DPBS and filter through 0.22 µm filter. Make 1.0 ml aliquots and store at -20˚C.
200 ml DPBS
3g BSA
3g trypsin inhibitor
Hi Ovomucoid (6X)
To 150 ml DPBS (Thermo Fisher Scientific, 14287-080), add 6 g BSA (Sigma A8806). Add 6 g Trypsin inhibitor (Worthington, LS003083) and mix to dissolve. Adjust pH to 7.4; requires the addition of at least 1.5ml of 1N NaOH. When completely dissolved, filter through 0.22 µm filter. Make 1.0 ml aliquots and store at -20˚C.
Insulin (0.5 mg/ml; 100X)
To 20 ml sterile water add 10 mg insulin (Sigma, I-6634), 100ul 1.0 N HCl. Mix well. Filter through 0.22µm filter. Store at 4˚C for 4 to 6 weeks.
NAC (5 mg/ml; 1000X)
Dissolve 50 mg N-acetyl cysteine (Sigma A8199) in 10 ml Neurobasal (Gibco, 21103-049) (will be yellowish). Filter through 0.22 µm filter and make 500 µl aliquots; store at -20˚C.
Poly D-Lysine Water (PDL) (100X)
Add 5 ml ddH2O in a 5 mg bottle of PDL (Sigma P6407). Filter through 0.22µm filter. Make 100 µl aliquots; store at -20˚C.
DNase stock solution - Required concentration for the primary cell isolation: 12500Unit/mL
Use DNaseI (Worthington, LS002007, 100 mg). EBSS (Gibco, 14155-063-500ml; Free of CaCl2, MgCl2 and PhenolRed). Calculate the amount of 1X EBSS to dissolve DNase1 to get 12500Unit/ml. Once dissolved, Filter through 0.22µm filter. Make 200ul Aliquot. store -20˚C.
Papain - Worthington Biochemicals, LK003176
DPBS (Dulbecco’s Phosphate Buffered Saline) – (Gibco, 14287-080)
DMEM (Gibco, 11960-044)
HI-FBS (Gibco, 10437-028)
Penicillin-Streptomycin (Pen/strep) (10,000 U/mL) (Gibco, 15140148)
L-Glutamine (GIBCO 25030)
Sodium Pyruvate (GIBCO 11360)
Trypsin (0,05% Trypsin-EDTA) (Gibco, 25300-054)
Normal Goat Serum (NGS) - (ThermoFisher 01-6201)
Astrocyte Growth Media (AGM)
Astrocyte Growth Media (AGM)
This recipe makes 500ml of filtered astrocyte growth media. Store in tissue culture fridge after use.
Wash filter with a bit of DMEM and vacuum off.
To the filter holder, add the following media components:
Component Volume Location in lab
DMEM (#11960) 450ml TC fridge
HI*-FBS 50ml TC freezer
Penstrep (100x) 5ml TC freezer
L-Glutamine (100x) 5ml TC freezer (use a fresh aliquot)
Sodium Pyruvate (100x) 5ml TC fridge
Insulin 5ml TC fridge
N-Acetyl Cysteine 500µl TC freezer – labelled NAC
Hydrocortisone 500µl TC freezer – labelled H
*Heat Inactivated
Filter vacuum and store in tissue culture fridge.
Astrocyte Culture
Astrocyte Culture
PREP DAY = DIV0 (Days in vitro 0)
Take prep DPBS (Cat #14287-080) from the cold room (Don’t need to add Phenol red for only astrocytes).
Preparation of Culture Flasks for Astrocytes
SPRAY DOWN EVERYTHING IN THE TISSUE CULTURE HOOD. Take out fresh flask of DPBS from cold room and add phenol red (1:1000).
Dilute 100µl PDL (poly-D-lysine--see Materials) aliquots stored in -20°C in 10ml water. Add 10ml to each flask.
Coat 75cm² culture flasks with 10 µg/ml PDL for at least 30 mins at room temperature.
Wash 3 times with sterile ddH2O, then add 15ml AGM to each flask. Let sit in incubator with lid slightly twisted off during prep to let the CO2 levels and temperature equilibrate.
Preparation of Solutions for Astrocytes for 9 Pups
Prepare the following solutions:
| A | B | C | D | |
| Tube Label | DPBS | # of Aliquots + Additive | Notes | |
| Lo OVO | 9mL | 1 1mL aliquot | Add 100µL DNase before use | |
| Hi OVO | 6mL | 1 1mL aliquot |
Warm 90mL AGM (for 1 litter) in the water bath.
For each litter – prepare 2 universal tubes with papain resuspended in PBS. Each vial gets 20ml PBS, 2 vials of papain, and 800µl DNaseI (add DNaseI right before adding tissue). Place in the water bath to warm. You will finish preparing the papain near the end of the dissection.
Papain = gentler form of trypsin. To prepare, add 10mL of prewarmed DPBS to papain vial (stored at -20C). Dissolve and return to universal tube. Remember to add 800µl DNase (stored at -20C) per universal tube just before adding the tissue.
Mouse Brain Dissection
Mouse Brain Dissection
Prepare dissection surface by cleaning with ethanol, placing an absorbent pad on the benchtop, and preparing tools. Will need small curved spring scissors, larger scissors for decapitation, and forceps.
Use tools ONLY in tissue culture room. Tools should also be rinsed with water – NEVER use detergent.
Prepare 4 60mm dishes, adding 6ml of DPBS into each dish. Each dish would contain 3 brains, and one dish is used for dissection.
Decapitate pups and de-skin the heads. Remove skull using small scissors to trim around but above the ears up to the midline. Skull should peel off.
Remove the brains and place into a 60mm dish with DPBS.
Cut off the cerebella. Remove basal ganglia by trimming axons and scooping out the tissue. Remove meninges from both surfaces of the brain using forceps. Remove hippocampus from both hemispheres. This will leave isolated cortex.
Transfer cortices to a fresh 60mm dish with DPBS.
Chop the cortices with sterile razorblade such that each tissue chunk is less than 1mm³.
Tissue Digestion
Tissue Digestion
Resuspend the papain. And add the DNase.
Cut off the tip of sterile plastic pipette to make a larger opening.
Using the plastic pipette, suck up the chopped tissue and DPBS. Tap the tip of the pipette onto the papain solution in 20 ml universal tubes so tissue settles towards bottom of tip, then add to tube, minimizing amount of liquid transferred. Swirl gently to get a ‘tornado effect’ before letting the brain tissue settle. Add half of the tissue to each of the tubes (ideal would correspond to 5 brains / tube for mouse).
Incubate at 33°C for 45 minutes, swirling the tissue at 15-minute intervals.
Never above 34°C – papain is temperature sensitive. Do NOT swirl at the 45min time-point.
Just before the end of the papain incubation, add DNase to the Lo Ovo (400µl).
Aspirate off the supernatant with a fresh vacuum pipet. Be sure to suck off the bubbles first and take care not to aspirate tissue. Try to remove as much media as possible.
Add 2 ml Lo-Ovo gently by dribbling down the side of the tube. Triturate using a 1ml pipette about 10 passes, starting out slowly and then speeding up. Let the tissue settle about 90 seconds. Check that there is a suspension, otherwise, 1ml of Lo-Ovo can be added to help in resuspension. Add the remainder of Lo-Ovo to dissociated cells and move to a 50ml conical tube. You can combine tubes at this point.
Ovo breaks up tissue into single cells and inactivates papain. Aspirate 1ml and release gently in a circular manner around the vial.
Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
This pellet is often fragile, can go up to 1400rpm for spin.
Aspirate off the supernatant with vacuum.
Very delicate and important step affecting yield. Do not aspirate all solution, ~1ml of media can be left to avoid disturbing pellet when aspirating supernatant.
Resuspend the cells in 2 ml Hi-Ovo. Triturate using a 1ml pipette about 10 passes. Add the remainder of Hi-Ovo.
Cells do not thrive well in Hi-Ovo so be sure to finish step 9 under 2 minutes.
Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
Aspirate off the supernatant with vacuum. Here the protocol splits in two for neurons and astrocytes.
Astrocyte Prep
Astrocyte Prep
Resuspend the cells in 2-3ml warmed AGM. Bring up to 10ml. Filter the cell mixture through 100µm strainer 1 mL at a time into a 50ml Falcon tube. Remember to Ethanol/flame sterilize forceps. It also helps to wet the filter by stabbing it into the tube with a P1000 pipet with 1ml of AGM.
Centrifuge the filtered cells at 900rpm for 9 minutes at room temperature.
Aspirate off the supernatant and resuspend the cells in 2 mL AGM.
Bring up the volume to 10ml to count cells. Can pool cells from multiple universal tubes if necessary.
Count the cells. Make sure to zoom in to check. Plate cells at very high density: 30 million cells per 75cm² culture flask as minimum. Better if can be between 40-45 million. Add the required amount to the prepped flasks.
Store in incubator with cap slightly unscrewed.
Roughly 3 pups will yield ~40 million cells, plate in a T75 flask.
Astrocyte Shake-off (DIV3)
Astrocyte Shake-off (DIV3)
Warm up AGM and DPBS in water bath.
Check astrocytes for normal morphology under microscope.
Wash in 10ml DPBS at least 3 times.
Add 20ml of DPBS and shake hard 8 times on each hand (amount of shaking needs to be determined on an individual basis).
Aspirate the DPBS and dislodged cells.
Add another 20ml of DPBS. Check under microscope to see if cell debris and other cells have been dislodged. There should be patches of ‘holes’ for which the astrocytes can grow into. If insufficient, shake more/harder.
Add 15ml of AGM to the flask.
Store in incubator.
Feed Astrocytes with AraC (DIV5)
Feed Astrocytes with AraC (DIV5)
Add AraC (cytosine arabinoside) to AGM at 1:1000. Warm in incubator. [AraC stock is 10mM].
Replace half of the media with the AraC media (final concentration of AraC = 1:2000).
Passage Astrocytes (DIV 7) – Includes PDL Coating on Plates
Passage Astrocytes (DIV 7) – Includes PDL Coating on Plates
Warm up trypsin, AGM (2 tubes of 15mL in 50mL tubes and 1 tube of 5mL in 15mL tube) and DPBS in water bath.
Prepare the plates in which astrocytes will be passaged into by coating wells with PDL.
Remove old media in flask.
Rinse flask with DPBS 3 times.
Add 10 mL pre-warmed trypsin to each flask. Incubate for 6-7mins in 37°C incubator, lying flat (not stacked) to have even heat.
Tap flask to loosen astrocytes and rinse off with 10mL pipette to ensure all of the astrocytes are dislodged. Pool cells from 3 flasks to 2 tubes (each 15mL of cells + trypsin to the prepared 2 tubes of 15mL AGM).
Centrifuge at 1.1 x 1000RPM for 10mins to pellet astrocytes.
Vacuum off the supernatant.
Resuspend gently each tube in 2mL of AGM. Pipette up and swirl to dislodge the pellet. Dispense and swirl gently until a cell suspension is achieved. Combine both tubes and add an additional 1mL of AGM to bring up final volume to 5mL.
Take 10µL of cell suspension for cell counting (add 10µL of Tryphan blue to stain cells).
Use ‘Rat astrocyte protocol’ on Countess to expose cells such that they are bright in the middle.
Take the average of both live cell readings and take note of cell viability (>90%).
Ideally, each flask is to yield 3-4 million cells. >4 million astrocytes may suggest that the shake off was not able to remove all the cell types, and may be contaminated i.e. with fibroblasts.
Passage the following amounts:
- 6 well plate: 300,000 – 400,000 cells/well
- 12 well plate: 150,000 – 200,000 cells/well
- 10cm plate: ~3 million cells
Add the appropriate number of astrocytes into each well.
Acknowledgements
This protocol was adapted from the Eroglu lab for use in the Gersbach lab.