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Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during immune reaction. 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] Microglia Cultures and Mixed Glial Culture

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, 9869 views, 3 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.149

[Abstract] Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during immune reaction. 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, InvitrogenTM, catalog number: 11330-032)
  3. D-Glucose
  4. Sterile water
  5. Sterile PBS
  6. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044)
  7. Non-essential amino acids (100 ml) (Life Technologies, Gibco®, catalog number: 11140-050)
  8. Sodium pyruvate (100 ml) (Sigma-Aldrich, catalog number: S8636)
  9. 200 mM L-glutamine (100 ml) (Life Technologies, Gibco®, catalog number: 25030-081)
  10. Penicillin/streptomycin (100 ml) (Sigma-Aldrich, catalog number: P0781)
  11. DMEM/F12-based culture medium (see Recipes)
  12. Poly-D-lysine stock solution (see Recipes)
  13. Treatment medium (see Recipes)

Equipment

  1. Cell culture incubator
  2. Centrifuges
  3. Dissection microscope
  4. Scissors and forceps
  5. T175 flasks
  6. 70 µm nylon filter (sterile)
  7. 50 ml tube
  8. Sterile filter (0.2 µm)
  9. Foil
  10. Laminar hood

Procedure

  1. Seed cells from 2-2.5 rat brains or 4-5 mouse brains from pups of postnatal day 1 to 5 in to one T175 flask.
  2. Coat T175 flasks with 20 ml Poly-D-lysine (20 µg ml-1) in a laminar hood for 2-3 h or in the in incubator for at least 1 h.
  3. Wash the flasks twice with 25 ml of sterile water. Add 25 ml sterile PBS to each flask.
  4. the animal procedure room, remove whole brains from 1-3 day-old rat or mouse pups and place them in cold DMEM/F12.
  5. Under a microscope, remove olfactory bulb and cerebellum from the brain. Remove meninges and blood vessels.
  6. Pool tissues from up to 10 brains into a 6 cm petri dish and keep dish on ice.
  7. 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.
  8. Filter the minced tissue suspension through a 70 µm nylon filter and centrifuge the filtrate for 10 min at 6.5x speed setting (~1,500 rpm).
  9. Carefully remove the supernatant using 10 ml pipet and resuspend the pelleted cells in 10-20 ml of DMEM/F12-based culture medium.
  10. Completely remove the PBS from flasks. Add 25 ml of pre-warmed (37 °C) DMEM/F12-based culture medium to each flask.
  11. Resuspend the cell suspension again and put the cells to each flask preloaded with medium. Place the flasks into a humidified 37 °C incubator with 5% CO2.
  12. Four days after the initial seeding, remove the medium and add 25 ml of fresh warm culture medium to each flask.
  13. Mixed glia cultures will be ready for shaking off microglia at between 12-14 days after initial seeding.
  14. Separate microglia from astrocytes by shaking the flasks for 5 h at 180 rpm. The enriched microglia are >98% pure as determined by OX-42-immunostaining and glial fibrillary acidic protein (GFAP)-immunostaining.

Recipes

  1. Poly-D-lysine solution
    Dissolve in 50 ml of ddH2O to make 5x stock solution.
    Dilute with sterile ddH2O to 20 µg/ml right before use.
  2. DMEM/F12-based culture medium
    Reagents
    Volume
    Final con.
    DMEM/F12
    430 ml
    -
    Heat-inactivated fetal bovine serum*
    50 ml
    10%
    None essential nonessential amino acids
    5 ml
    0.1 mM
    Sodium pyruvate
    5 ml
    1 mM
    L-glutamine
    5 ml
    2 mM
    Penicillin/streptomycin
    5 ml
    50 U/ml/50 µg/ml
    on.: 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. Treatment medium
    Reagents
    Volume
    Final con.
    DMEM/F12
    470 ml
    -
    Heat-inactivated fetal bovine serum*
    10 ml
    2%
    None essential nonessential amino acids
    5 ml
    0.1 mM
    Sodium pyruvate
    5 ml
    1 mM
    L-glutamine
    5 ml
    2 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. 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.


How to cite: Gao, H. (2011). Microglia Cultures and Mixed Glial Culture. Bio-protocol Bio101: e149. DOI: 10.21769/BioProtoc.149; Full Text



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3/10/2014 6:31:30 PM  

Yoojin Seo
Seoul national univ.

Hi, thank you for your kind instruction. I followed yours but i cannot get enough microglial cells... How many cells can you usually get using shaking methods? I seeded whole cells from 1 neonate mouse onto 75 T flask and maintain culture for 14 days....

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4/6/2012 12:05:55 PM  

Is it M-CSF or GM-CSF necessary for the microglia culture?

4/7/2012 1:43:51 PM  

Huiming Gao (Author)
Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park

M-CSF and GM-CSF have been found to induce microglial proliferation in fetal and adult cultures. However, we have never added exogenous M-CSF or GM-CSF into our culture medium. It is possible that low levels of M-CSF and/or GM-CSF in fetal bovine serum may help microglia cultures grow.

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8/15/2011 3:38:37 PM  

Anonymous Lin
Test institute

explan i n brief plz,......
Coat T175 flasks with 20 ml Poly-D-lysine (20 µg/ml) in a laminar hood for 2-3 hr or in the in incubator for at least 1 hour.

regards

robin.hood008@yahoo.com

8/31/2011 4:04:51 PM  

bio-protocol

For the sterile purpose, in a laminar hood, add 20 ml of 20 µg/ml Poly-D-lysine into T175 flasks. leave the flasks in the laminar hood for 2-3 hrs or put the flasks into incubator for 1 hour. then remove the Poly-D-lysine from flasks and wash the flasks as described.

Reply

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