Apr 20, 2026
Astrocyte Isolation and Transfection for Astrocytes/Neuron co-culture assay
Forked from Astrocyte Isolation and ACM Production
- Justin T Savage1,
- Juan Ramirez1,
- Dolores Irala2,
- Cagla Eroglu1,2,3,
- Sarah van Veen4,5
- 1Department of Neurobiology, Duke University Medical Center, Durham, NC 27710;
- 2Department of Cell Biology, Duke University Medical Center, Durham, NC 27710;
- 3Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710;
- 4KU Leuven;
- 5Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
External link: https://doi.org/10.1016/j.crmeth.2024.100861
Protocol Citation: Justin T Savage, Juan Ramirez, Dolores Irala, Cagla Eroglu, Sarah van Veen 2026. Astrocyte Isolation and Transfection for Astrocytes/Neuron co-culture assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dy7kvqe/v1
Manuscript citation:
Savage JT, Ramirez JJ, Risher WC, Wang Y, Irala D, Eroglu C (2024) SynBot is an open-source image analysis software for automated quantification of synapses. Cell Reports Methods 4(9). doi: 10.1016/j.crmeth.2024.100861
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: February 03, 2025
Last Modified: April 26, 2026
Protocol Integer ID: 119472
Keywords: rat cortical astrocyte, cortical astrocyte, transfection for astrocyte, astrocyte isolation, astrocyte, synaptogenesis assay, media for synaptogenesis assay
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative Grant
Grant ID: ASAP-020607
Abstract
Protocol for isolating rat cortical astrocytes and producing astrocyte conditioned media for synaptogenesis assays.
Materials
STOCK SOLUTIONS:
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 sterilize the solution by filtration.
Store at -20 °C and protect from light.
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 approx. 1ml of 1N NaOH.
When completely dissolved bring to 200 ml DPBS, filter through 0.22 µm filter.
Make 1.0 ml aliquots and store at -20˚C.
Hi Ovomucoid (6X)
To 150 mls 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 -GP filter.
Make 1.0 ml aliquots and 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 prep: 12500Unit/mL
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.
Insulin – (Sigma I1882) - 10mg of insulin in 20ml of TC water. Vortex to make sure that the powder dissolves well before filtering.
Syringe Filter 0.22 µm – Millipore SLGPR33RB
Nitex filter - filter 3-20/14 from Tetko, Inc. Cut into 3-inch squares, wrap in small packets of foil, and autoclave.
Papain - Worthington Biochemicals, LK003176
DPBS (Dulbecco’s Phosphate Buffered Saline) – (Gibco, 14287-080)
DMEM – (Gibco, 11960044)
FBS – (Gibco, 10437028)
Penicillin-Streptomycin (10,000 U/mL) (Gibco, 15140148)
L-Glutamine(GIBCO 25030)
Sodium Pyruvate (GIBCO 11360)
Trypsin (0,05% Trypsin-EDTA) (Gibco, 25300-054)
Vivaspin tube (Sartorius, Cat. VS2012. MWCO 5000; 20 mL tubes)
Low protein binding tubes (Eppendorf 022431081)
Minimal media (Neurobasal medium minus phenol red, 100 U/mL Pen/Strep, 2 mM L-Glutamine, 1 mM Na Pyruvate
Cortical Astrocyte Isolation (Prep Day)
Prepare the following solutions (enough for 9 pups)
| Tube Label | DPBS | # of Aliquots + Additive | Notes | |
| Lo OVO | 36ml | 2 2ml aliquot | Add 400µl DNase before use | |
| Hi OVO | 20ml | 2 2ml aliquot | ||
| BSA Buffer | 76ml | 2 2ml aliquot | Divide among 2 50ml Falcon Tubes | |
| Panning (X2 tubes) | 45ml | 500µl insulin + 5ml BSA buffer | Insulin, do not use if >6 weeks’ old |
Prepare Astrocyte Growth Media (AGM)
This recipe makes 500ml of filtered astrocyte growth media. Store at 4C.
Combine and filter the following reagents:
- DMEM 450ml
- FBS 50ml
- Penstrep (100x) - 5ml
- Glutamate (100x) - 5ml
- Sodium Pyruvate (100x) - 5ml
- Insulin - 5ml
- N-Acetyl Cysteine - 500µl
- Hydrocortisone - 500µl
Preparation of culture flasks
Dilute 100µl of PDL in 10ml water.
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 the incubator with the lid slightly twisted off during prep to let the CO2 levels and temperature equilibrate.
Rat 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.
Prepare 3 60mm dishes adding 5ml of DPBS to collect 6 cortices each, and one 10cm dish with 12ml of DPBS for the dissection.
Decapitate pups and de-skin the heads. Remove the skull using small scissors to trim around but above the ears up to the midline. The skull should peel off.
Remove the brains and place them into the 10cm dish with DPBS.
Using the forces remove meninges and dissect the basal ganglia, hippocampus and the cerebella. This will leave isolated cortex.
Transfer cortices to a fresh 60mm dish with DPBS.
Chop the cortices with scissors to increase the surface for enzymatic digestion.
Tissue digestion
Resuspend the papain. And add the DNase.
Cut off the tip of sterile plastic disposable pipette to make a larger opening.
Using the plastic pipette, suck up the chopped tissue and DPBS. Tap the tip of the pipet onto the papain solution in 20 ml universal tubes without squeezing the pipet or adding liquid. Swirl gently to get a ‘tornado effect’ before letting the brain tissue settle
Incubate at 33°C for 45 minutes, swirling the tissue at 15-minute intervals.
Just before the end of the papain incubation, add DNase to the Lo Ovo (400ul).
Once the 45-minute incubation is completed, aspirate the DPBS from the universal tubes. The tissue will stay at the bottom of the tube.
Add 2 ml Lo-Ovo gently by dribbling down the side of the tube. Triturate using a 1ml pipette about 10 passes up and down. Check that there is a single cell suspension, otherwise, 1ml of Lo-Ovo can be added to help in resuspension. Add the remainder of Lo-Ovo to dissociated cells.
Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
Aspirate off the supernatant with a vacuum without disturbing the pellet.
Resuspend the cells in 2 ml Hi-Ovo. Triturate using a 1ml pipette about 10 passes. Add the remainder of Hi-Ovo.
Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
Aspirate off the supernatant with a vacuum.
Resuspend the cells in 2-3ml warmed AGM. Bring up to 10ml. Filter the cell mixture through 20m Nitex membrane 1 mL at a time into a 50ml Falcon tube. Remember to Ethanol/flame sterilize forceps.
Centrifuge the filtered cells at 1100rpm for 10 minutes at room temperature.
Aspirate off the supernatant and resuspend the cells in 2 mL AGM.
Bring up the volume to 10ml to count the cells.
Plate cells at very high density: 15 million cells per 75cm2 culture flask as a minimum. Better if can be between 17-20 millon. Add the required amount to the prepped flasks.
Store in incubator with cap slightly unscrewed.
DIV 3 Astrocyte Shake-off
Warm-up AGM and DPBS in a water bath
Check astrocytes for normal morphology under a microscope
Wash the cells with 10ml DPBS at least 3 times
Add 15ml of DPBS and shake the flasks at least 10 times (this allows for other cell types to detach from the coating).
Aspirate the DPBS and dislodged cells
Add another 15ml of DPBS. Check under a microscope to see if cell debris and other cells have been dislodged. There should be patches of ‘holes’ into which the astrocytes can grow into. If insufficient, shake more/harder.
Add 15ml of AGM
Store in incubator
DIV 5 Feed Astrocytes with AraC
Warm-up AGM in a water bath and add AraC (1:10000)
Check astrocytes for normal morphology under a microscope
Remove old AGM and replace with fresh AGM+AraC (15ml/flask)
Store in the incubator
DIV 7 Astrocyte Passage
Note
This protocol is for 1 flask of astrocytes. Scale up accordingly.
Warm-up trypsin, AGM and DPBS in a water bath
Dilute 100µl of PDL in 10ml water and coat the wells of a 12-well plate with 0.5 ml of 10 µg/ml PDL for at least 30 min at room temperature.
Wash 3 times with sterile ddH2O, then add 1 ml AGM to every well. Let sit in the incubator during the astrocyte passaging to let the CO2 levels and temperature equilibrate.
Remove old AGM media in the flask
Rinse the flask with PBS without magnesium and calcium 3 times
Add 10 ml pre-warmed trypsin to each flask. Incubate for 5 minutes at 37˚C in the incubator, lying flat (not stacked) to have even heat.
Tap flask to loosen astrocytes and collect with 10ml pipette all the astrocytes that are now are dislodged.
Centrifuge at 1.1 x 1000RPM for 10min to pellet the astrocytes
Vacuum off the supernatant
Resuspend the pellet in 2ml of AGM. Pipette up and swirl to dislodge the pellet. Dispense and swirl gently until a cell suspension is achieved.
Plate the astrocytes in the 12-well plate at 0.2 million cells per well.
Store in the incubator.
DIV8 Astrocyte Transfection
Warm up an aliquot of AGM in incubator.
To each well in a 12-well, the following components are added:
DNA - 1 ul of 1 ug/ul
Plus - 1 ul
Optimem - 64 ul
Mix all 3 components and let set for 5 min.
To another tube, prepare a LTX mixture tube: 2 ul LTX + 64 ul Optimem/condition tested.
Add 66 ul LTX mixture to each transfection tube and incubate for 30 min at room temperature.
Remove and save 0.5 ml of old AGM. Add 0.5 ml of fresh AGM to the astrocytes and another 0.5 ml of fresh AGM to the saved media.
Add 132 ul of DNA/LTX to each well in a swirl fashion.
Incubate 2.5h. Warm up an aliquot of DPBS.
Wash 3 times with 1 ml of DPBS.
Add the 1 ml of saved/fresh AGM mixture to each well.
DIV10 Co-culture astrocytes with neurons
Warm up trypsin, DPBS and 10 ml aliquot of AGM.
Aspirate old AGM and wash 3 times with DPBS.
Add trypsin (0.5 ml per well for 12-well plate). Incubate for 6 min at 37°C.
Add 1 ml of AGM into fresh 15 ml tube per condition.
Loosen the astrocytes by tapping gently on the plate after incubation. Transfer into prepared 15 ml tube.
Centrifuge at 1.1 x 1000RPM for 10min to pellet the astrocytes
Remove medium and resuspend in neuronal medium.
Count and plate 20000 astrocytes to each well of neurons (for neuron isolation protocol, see protocol below).