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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, 7079 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.

材料和试剂

  1. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P7280)
  2. MEM(Life Technologies,Gibco ,目录号:11090-08)
  3. D-葡萄糖
  4. 无菌水
  5. 无菌PBS
  6. 台盼蓝染料
  7. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000-044)
  8. 热灭活的马血清(HS)(Life Technologies,Gibco ,目录号:26050-088)
  9. 无必需的非必需氨基酸(Life Technologies,Gibco ,目录号:11140-050)(100ml)
  10. 丙酮酸钠(Sigma-Aldrich,目录号:S8636)(100ml)
  11. 200mM L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)(100ml)
  12. 青霉素/链霉素(Sigma-Aldrich,目录号:P0781)(100ml)
  13. 聚D-赖氨酸储备溶液(见配方)
  14. 维护培养基(参见配方)
  15. 处理介质(见配方)

设备

  1. 细胞培养孵化器
  2. 标准台式离心机
  3. 血细胞计数器
  4. 解剖显微镜
  5. 剪刀和镊子
  6. 无菌过滤器(0.2μm)

  7. 24孔板
  8. 层流罩
  9. 50 ml管
  10. 10 ml移液器

程序

  1. 包衣和洗涤培养板
    1. 在层流罩中,用无菌水稀释聚D-赖氨酸储备溶液(5x)至20μg/ml。
    2. 向24孔板的每个孔中加入0.25ml。
    3. 离开板在敞篷2-3小时或在孵化器至少1小时。
    4. 使用前,取下涂层溶液。
    5. 用1ml /孔的无菌水洗孔两次。
    6. 向每个孔中加入1ml无菌PBS。 在使用前彻底清除PBS。
  2. 在动物手术室中,从胚胎期13/14时的怀孕大鼠或小鼠中取出胚胎,并将胚胎置于冷MEM中。
  3. 在显微镜下,解剖出胚胎大鼠或小鼠大脑的中脑部分。 去除脑膜和血管。 池组织并保持在冰冷的MEM
  4. 在层流罩,转移组织到50ml管。 轻轻研磨组织(每次5-10次),首先用10 ml移液管,然后用1 ml移液管吸头安装到10 ml移液管,然后用一个适合的200μl移液器吸头。
  5. 以6.5倍速度设定(〜1,500rpm)将研磨的组织离心10分钟
  6. 小心地取出上清液,并将沉淀的细胞重悬在10ml维持培养基中
  7. 取30微升的细胞悬液,并与270微升的台盼蓝染料混合。 加载10微升到血细胞计数器以计数细胞密度
  8. 用维持培养基将细胞密度调节至1×10 6个细胞/ml。
  9. 向聚-D-赖氨酸包被的24孔板的每个孔中加入0.5ml细胞。
  10. 将板放置在具有5%CO 2的湿润的37℃培养箱中
  11. 初始接种后三天,向每个孔中加入0.5ml温热(37℃)维持培养基
  12. 在初始接种后7天,培养物将准备用载体或所需试剂在处理培养基中处理
  13. 在治疗时,神经胶质细胞培养物由〜10%小胶质细胞,50%星形胶质细胞和40%神经元组成,其中2-3%是酪氨酸羟化酶 - 免疫反应性神经元。

食谱

  1. 聚-D-赖氨酸溶液
    溶解在50ml ddH 2 O中以制备5x储备溶液。
    保持为5.0毫升等分在-20°C。
    在使用前用无菌ddH 2 O稀释。
  2. 维持培养基
    试剂
    音量
    final con。
    MEM
    380 ml
    -
    D-葡萄糖
    0.5克
    1 g/L
    热灭活的胎牛血清*
    50 ml
    10%
    热灭活的马血清**
    50 ml
    10%
    无必需的非必需氨基酸
    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
    50U/ml /50μg/ml
    浓度:
    无菌过滤器(0.2μm),存放在4°C的箔中 *在56℃热灭活30分钟,并以50ml等分试样储存于-70℃ **在-20℃下以50ml等分试样储存
  3. 处理介质
    试剂  
    音量
    最终结果
    MEM
    465毫升
    -
    热灭活的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
    50U/ml /50μg/ml
    无菌过滤器(0.2μm),存放在4°C的箔中

致谢

这个协议已经由Hong的实验室中的各种研究者,特别是Liu Liu博士(Ga> et al。,2002; Liu et al。 ,2000)。

参考文献

  1. Gao,H.M.,Hong,J.S.,Zhang,W.and Liu,B。(2002)。 小胶质细胞在鱼藤酮诱导的多巴胺能神经元变性中的独特作用 Neurosci 22(3):782-790。
  2. Liu,B.,Du,L.and Hong,J.S。(2000)。 纳洛酮通过抑制小胶质细胞活化和超氧化物生成来保护大鼠多巴胺能神经元免受炎症损伤。 J Pharmacol Exp Ther 293(2):607-617。
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How to cite this protocol: 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; Full Text
  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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    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.

    Reply

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