Midbrain Neuron-glia Mixed Cultures   

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Original research article

A brief version of this protocol appeared in:
The Journal of Neuroscience
Feb 2002


Mixed neuron-glia cultures provide a unique tool to study cellular contribution and molecular pathways in various neurological disorders. They are also invaluable for exploring neuron-glia interaction under physiological and pathological conditions. The relatively long-lasting midbrain neuron-glia mixed cultures generated following this protocol have been widely used to study the pathogenesis of Parkinson’s disease, the most common neurodegenerative movement disorder.

Materials and Reagents

  1. Poly-D-lysine (Sigma-Aldrich, catalog number: P7280 )
  2. MEM (Life Technologies, Gibco®, catalog number: 11090-08 )
  3. D-Glucose
  4. Sterile water
  5. Sterile PBS
  6. Trypan blue dye
  7. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
  8. Heat-inactivated horse serum (HS) (Life Technologies, Gibco®, catalog number: 26050-088 )
  9. None essential nonessential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 ) (100 ml)
  10. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 ) (100 ml)
  11. 200 mM L-glutamine (Life Technologies, Gibco®, catalog number: 25030-081 ) (100 ml)
  12. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P0781 ) (100 ml)
  13. Poly-D-lysine stock solution (see Recipes)
  14. Maintenance culture medium (see Recipes)
  15. Treatment medium (see Recipes)


  1. Cell culture incubator
  2. Standard benchtop centrifuges
  3. Hemocytometer
  4. Dissection microscope
  5. Scissors and forceps
  6. Sterile filter (0.2 µm)
  7. Foil
  8. 24-well plates
  9. Laminar hood
  10. 50-ml tube
  11. 10-ml pipet


  1. Coating and washing culture plates
    1. In a laminar hood, dilute poly-D-lysine stock solution (5x) with sterile water to 20 µg/ml.
    2. Add 0.25 ml to each well of 24-well plates.
    3. Leave the plates in the hood for 2-3 h or in the in incubator for at least 1 h.
    4. Before use, remove the coating solution.
    5. Wash the wells twice with 1 ml/well of sterile water.
    6. Add 1 ml sterile PBS to each well. Completely remove the PBS right before use.
  2. In the animal procedure room, remove embryos from time-pregnant rats or mice at embryonic day 13/14 and place embryos in cold MEM.
  3. Under a microscope, dissect out the midbrain portion of the embryonic rat or mouse brains. Remove meninges and blood vessels. Pool tissues and keep in ice cold MEM.
  4. In a laminar hood, transfer tissues to a 50-ml tube. Gently triturate the tissues (5-10 times each) first with a 10-ml pipet, then with a 1-ml pipet tip fitted to the 10-ml pipet followed by a fitted 200 µl pipet tip.
  5. Centrifuge the triturated tissues for 10 min at 6.5x speed setting (~1,500 rpm).
  6. Carefully remove the supernatant and resuspend the pelleted cells in 10 ml of maintenance culture medium.
  7. Take 30 µl of the cell suspension and mix with 270 µl of Trypan blue dye. Load 10 µl onto a hemocytometer to count cell density.
  8. Adjust the cell density to1 x 106 cells/ml with maintenance culture medium.
  9. Add 0.5 ml of cells to each well of the poly-D-lysine-coated 24-well plate.
  10. Place the plates in a humidified 37 °C incubator with 5% CO2.
  11. Three days after the initial seeding, add 0.5 ml of warm (37 °C) maintenance culture medium to each well.
  12. Seven days after initial seeding, cultures will be ready for treatment with vehicle or desirable reagents in treatment medium.
  13. At the time of treatment, the neuron-glia cultures are made up of ~10% microglia, 50% astrocytes, and 40% neurons of which 2-3% are tyrosine hydroxylase-immunoreactive neurons.


  1. Poly-D-lysine solution
    Dissolve in 50 ml of ddH2O to make 5x stock solution.
    Keep as 5.0 ml aliquots at -20 °C.
    Dilute with sterile ddH2O right before use.
  2. Maintenance culture medium
    final con.
    380 ml
    0.5 g
    1 g/L
    Heat-inactivated fetal bovine serum *
    50 ml
    Heat-inactivated horse serum **
    50 ml
    None essential nonessential amino acids
    5 ml
    0.1 mM
    Sodium pyruvate
    5 ml
    1 mM
    5 ml
    2 mM
    5 ml
    50 U/ml/50 µg/ml
    Con.: concentration
    Sterile filter (0.2 µm) and store wrapped in foil at 4 °C.
    *Heat-inactivated at 56 °C for 30 min and stored in 50 ml aliquots at -70 °C.
    ** Stored in 50 ml aliquots at -20 °C.
  3. Treatment medium
    final con.
    465 ml
    Heat-inactivated FBS
    10 ml
    Heat-inactivated HS 
    10 ml
    Sodium pyruvate
    5 ml
    1 mM
    5 ml
    2 mM
    5 ml
    50 U/ml/50 µg/ml
    Sterile filter (0.2 µm) and store wrapped in foil at 4 °C.


This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu (Gao et al., 2002; Liu et al., 2000).


  1. Gao, H. M., Hong, J. S., Zhang, W. and Liu, B. (2002). Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 22(3): 782-790.
  2. Liu, B., Du, L. and Hong, J. S. (2000). Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J Pharmacol Exp Ther 293(2): 607-617.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gao, H. (2012). Midbrain Neuron-glia Mixed Cultures. Bio-protocol 2(7): e148. DOI: 10.21769/BioProtoc.148.
  2. Gao, H. M., Hong, J. S., Zhang, W. and Liu, B. (2002). Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 22(3): 782-790.

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sonia mazzitelli
manchester university
does anybody knows if i can separate neurones from mixed cultures?
10/16/2011 4:15:46 AM Reply
Huiming Gao
Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA

If you use transwell inserts to generate reconstituted cultures that contain neurons and glia, you can separate neurons from glia. Otherwise, it is impossible to separate neurons from glia in the mixed neuron-glia cultures. If you consider using transwells, I can upload my protocol for generating that type of reconstituted cultures.

1/6/2012 3:34:25 PM