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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.

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Midbrain Neuron-glia Mixed Cultures
中脑神经元-胶质细胞混合培养

神经科学 > 细胞机理 > 细胞分离和培养
作者: Huiming Gao
Huiming GaoAffiliation: National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
Vol 2, Iss 7, 4/5/2012, 6505 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.148

[Abstract] 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.

[Abstract] 混合神经元-神经胶质细胞培养提供了一个独特的工具来研究细胞的构成和各种神经系统疾病的分子途径。他们在探索生理和病理条件下神经元-胶质细胞相互作用方面大有用处。按照操作指南上相对持久的中脑的神经元—神经胶质细胞混合培养已被广泛用于研究帕金森氏症——最常见的神经性运动障碍疾病的发病机制。这份指南经过了Dr. Hong实验室不同研究人员尤其是Dr. Bin Liu多年的改进。

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)

Equipment

  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

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.
    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.

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.
    Dilute with sterile ddH2O right before use.
  2. Maintenance culture medium
    Reagents
    volume
    final con.
    MEM
    380 ml
    -
    D-Glucose
    0.5 g
    1 g/L
    Heat-inactivated fetal bovine serum *
    50 ml
    10%
    Heat-inactivated horse 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
    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
    Reagents  
    volume
    final con.
    MEM
    465 ml
    -
    Heat-inactivated FBS
    10 ml
    2%
    Heat-inactivated HS 
    10 ml
    2%
    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.

Acknowledgments

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).

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.

材料与试剂

 

1.          -D-赖氨酸 (Sigma P-7280)

2.          MEM  (Gibco 11090-08)

3.          D-葡萄糖

4.          热灭活胎牛血清 (FBS; Gibco 16000-044)

5.          热灭活马血清 (HS; Gibco 26050-088)

6.          不含非必须氨基酸(Gibco 11140-050; 100 ml)

7.          丙酮酸钠 (Sigma; S8636; 100 ml)

8.          L-谷氨酰胺(Gibco 25030-081; 100ml; 200 mM)

9.          青霉素/链霉素 (Sigma P0781; 100 ml)

 

仪器

 

1.          细胞培养孵育器

2.          离心机

3.          解剖镜

4.          剪刀和镊子

5.          无菌过滤器 (0.2 μm)

6.          铝箔

 

步骤

 

1.           涂布和清洗培养皿  

2.           在层流罩,用无菌水稀释聚-D -赖氨酸液(5X)至20μg/ml24孔板上每孔加入0.25 ml。至少1个小时,在2-3小时的油烟机离开或在孵化器的板块。将板放在层流罩中2-3hrs或在孵育器中至少1小时。

3.           使用前,除去图层溶液。约1 ml/孔的无菌水清洗2次。即将使用前彻底清除PBS

4.           在动物实验室中,清出怀孕大鼠或13/14日龄的小鼠胚胎并将胚胎放进预冷的MEM

5.           解剖镜下切除胚胎大鼠或小鼠的脑。除去脑膜和血管。将组织保存在冰上预冷的MEM中。

6.           在层流罩,将组织转移到一个50ml轻轻磨碎组织(每个5-10次)。先用10ml的移液管,然后依次用和其配套的1ml200 μl的枪头。

7.           6.5X转速(约1500 RPM)离心组织10min

8.           小心取出上清液,并重新悬浮在10ml传代培养基重悬颗粒细胞。

9.           30μL的细胞悬液和270μl台盼蓝染料液混合。 10μL到一个血球计数板以计算细胞密度。

10.       用传代培养基调整细胞密度1× 106细胞/ml-D -赖氨酸涂层24孔板上每孔加入0.5 ml细胞,然后置于5CO2 37℃保湿培养箱。

11.       初始培养3每孔0.5ml37预热的传代培养基

12.       初始培养7用合适的试剂配好培养基。

13.       在处理样品时,神经元-胶质细胞培养基由大约10%的小胶质细胞,50%的星形胶质细胞,和40%的神经元组成。40%的神经元中2-3%的酪氨酸羟化酶免疫神经元。

 

配方

 

1.         -D-赖氨酸:用50 mlddH2O溶解,配成 5X母液。分装成每管5.0 ml,保存在–20°C。使用前用灭菌ddH2O稀释。 

2.         传代培养基

试剂                                                                              体积                终浓度

MEM                                                                               380 ml                               -

D-葡萄糖                                                                        0.5 g                      1 g/l

热灭活胎牛血清*                                                             50 ml                    10%

热灭活马血清**                                                               50 ml                    10%

不含非必需氨基酸                                                           5 ml                     0.1 mM

丙酮酸钠                                                                          5 ml                     1 mM

L-谷氨酰胺                                                                      5 ml                     2 mM

青霉素/链霉素                                                                 5 ml                     50 U/ml/50 μg/ml

 

0.2 μm滤器过滤并用铝箔包裹储藏在 4°C

* 56°C热灭活 30 min并分装成每管50 ml,保存在 –70°C

** 50-ml分装并保存在 –20°C              

3.         处理培养基

试剂                                                                体积                    终浓度

MEM                                                               465 ml                -

热灭活 FBS                                                     10 ml                     2%

热灭活 HS                                                       10 ml                     2%

丙酮酸钠                                                           5 ml                     1 mM

L-谷氨酰胺                                                        5 ml                     2 mM

青霉素/链霉素                                                   5 ml                     50 U/ml/50 μg/mL

0.2 μm滤器过滤并用铝箔包裹保存在 4°C

 

参考文献

 

1.         Liu B., Du L., Hong J.S. (2000). Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. Journal of Pharmacology and Experimental Therapeutics 293(2): 607-17.

2.         Gao H.M., Hong J.S., Zhang W., Liu B. (2002). Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. Journal of Neuroscience 22(3): 782-90.  

 

 

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How to cite this protocol: Gao, H. (2012). Midbrain Neuron-glia Mixed Cultures. Bio-protocol 2(7): e148. DOI: 10.21769/BioProtoc.148; Full Text



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10/16/2011 4:15:46 AM  

sonia mazzitelli
manchester university

does anybody knows if i can separate neurones from mixed cultures?

1/6/2012 3:34:25 PM  

Huiming Gao (Author)
National Institute of Environmental Health Sciences, 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.

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