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Neuron-enriched cultures are a useful tool to study neuronal development and the molecular pathways that come in to play during neuronal death in various neurological disorders. This protocol is for generating midbrain neuronal cultures from late embryo rodent brain. This method uses Neurobasal medium and B27 serum-free supplement. The long-lasting (> 4 weeks) midbrain neuron-enriched cultures generated following this protocol have been wildly used to study the pathogenesis of Parkinson’s disease, the most common neurodegenerative movement disorder. The neuron-enriched cultures prepared following this protocol will contain < 10% astroglia. The usage of B27 serum-free supplement will definitively increase the cost. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

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[Bio101] Neuron-enriched Cultures (Method 2)

Neuroscience > Cellular mechanisms > Cell isolation and culture
Author: Huiming Gao
Huiming GaoAffiliation: Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
11/5/2011, 6036 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.150

[Abstract] Neuron-enriched cultures are a useful tool to study neuronal development and the molecular pathways that come in to play during neuronal death in various neurological disorders. This protocol is for generating midbrain neuronal cultures from late embryo rodent brain. This method uses Neurobasal medium and B27 serum-free supplement. The long-lasting (> 4 weeks) midbrain neuron-enriched cultures generated following this protocol have been wildly used to study the pathogenesis of Parkinson’s disease, the most common neurodegenerative movement disorder. The neuron-enriched cultures prepared following this protocol will contain < 10% astroglia. The usage of B27 serum-free supplement will definitively increase the cost. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

Materials and Reagents

  1. Poly-D-lysine (Sigma-Aldrich, catalog number: P7280)
  2. DMEM/F12 (Life Technologies, Gibco®, catalog number: 11330-032)
  3. D-Glucose (please add catalog number)
  4. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044)
  5. 200 mM L-glutamine (Life Technologies, Gibco®, catalog number: 25030-081) (100 ml)
  6. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P0781) (100 ml)
  7. Neurobasal medium (Life Technologies, InvitrogenTM, catalog number: 12348-017)
  8. 50x B27 serum-free supplement (Life Technologies InvitrogenTM/Gibco®, catalog number: 21103-049) (10 ml)
  9. Sterile PBS
  10. MEM
  11. Trypan blue dye
  12. Poly-D-lysine solution (see Recipes)
  13. Serum-containing culture medium (see Recipes)

Equipment

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

Procedure

  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-1. Add 0.25 ml to each well of 24-well plates. Leave the plates in the hood for 2-3 h or in the in incubator for at least 1 h.
    2. Before use, remove the coating solution. Wash the wells twice with 1 ml/well of sterile water. 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 for midbrain neuron-enriched cultures or at embryonic day 17/18 for cortical neuron-enriched cultures 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 10 ml pipet, then a 1 ml pipet tip fitted to the 10-ml pipet followed with 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 serum-containing 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 serum-containing culture medium. Add 0.5 ml of cells to each well of the poly-D-lysine-coated 24-well plate. Place the plates in a humidified 37 °C incubator with 5% CO2.
  9. Two days later, change cultures to 1 ml/well serum-free neurobasal medium.
  10. Treat seven-day-old cultures that contain less than 0.1% OX-42-immunoreactive microglia and about 3% glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes with desirable reagents or vehicle.

Recipes

  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.
  2. Serum-containing culture medium
    Reagents
    Volume
    Final con.
    DMEM/F12
    445 ml
    -
    D-Glucose
    3.0 g
    6 g/L
    Heat-inactivated fetal bovine serum*
    50 ml
    10%
    Penicillin/streptomycin
    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.
  3. Serum-free neurobasal medium
    Reagents
    Volume Final con.
    Neurobasal medium
    483.75 ml
    -
    B27 serum-free supplement
    10 ml
    1x
    L-glutamine
    1.25 ml
    0.5 mM
    Penicillin/streptomycin
    5 ml
    50 U/ml/50 µg/ml
    Sterile filter (0.2 µm) and store wrapped in foil at 4 °C.

References

  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. Lotharius, J., Dugan, L. L. and O'Malley, K. L. (1999). Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons. J Neurosci 19(4): 1284-1293.


How to cite: Gao, H. (2011). Neuron-enriched Cultures (Method 2). Bio-protocol Bio101: e150. DOI: 10.21769/BioProtoc.150; Full Text



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