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Primary culture of neurons from cerebral cortex is a popular model to study neuronal function in vitro and to explore the molecular mechanism of neurite outgrowth in the developing and adult central nervous system. This protocol is for preparing a culture of cerebral cortical neurons from postnatal rodent brain (Muramatsu et al., 2012). One day after cell plating, we can observe neurite outgrowth by microscope.

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Primary Culture of Cortical Neurons
皮层神经元的初代培养

神经科学 > 发育 > 神经元
作者: Rieko Muramatsu
Rieko MuramatsuAffiliation: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Japan
For correspondence: muramatsu@molneu.med.osaka-u.ac.jp
Bio-protocol author page: a566
 and Toshihide Yamashita
Toshihide YamashitaAffiliation: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Japan
For correspondence: yamashita@molneu.med.osaka-u.ac.jp
Bio-protocol author page: a379
Vol 3, Iss 8, 4/20/2013, 7441 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.496

[Abstract] Primary culture of neurons from cerebral cortex is a popular model to study neuronal function in vitro and to explore the molecular mechanism of neurite outgrowth in the developing and adult central nervous system. This protocol is for preparing a culture of cerebral cortical neurons from postnatal rodent brain (Muramatsu et al., 2012). One day after cell plating, we can observe neurite outgrowth by microscope.
Keywords: Brain(脑), Postnatal(产后), Mouse(鼠标), Vitro(体外), Dissociate(游离)

[Abstract]

Materials and Reagents

  1. Poly-L-lysine (Sigma-Aldrich, catalog number: P2636 )
  2. Dulbecco's Modified Eagle Medium, DMEM (Life Technologies, InvitrogenTM, catalog number: 12800-017 )
  3. Fetal bovine serum (Life Technologies, Gibco®, catalog number: 10437 )
  4. 2.5% Trypsin solution (Life Technologies, Gibco®, catalog number: 15090-046 )
  5. DNase (Sigma-Aldrich, catalog number: DN25 )
  6. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
  7. NaH2PO4.2H2O
  8. Na2HPO4.12H2O
  9. NaCl
  10. DMEM
  11. FBS
  12. Serum-containg culture medium (see Recipes)
  13. PBS (see Recipes)

Equipment

  1. Cell culture incubator
  2. Centrifuge
  3. Stereoscopic microscope
  4. Scissors, forceps, and knives
  5. Sterile filter (0.45 μm)
  6. Cell strainer 70 μm nylon (BD Biosciences, catalog number: 352350 )
  7. 24 well culture plate

Procedure

  1. Coating and washing culture plates.
    1. Dilute poly-L-lysine stock solution with sterile PBS to 100 μg/ml. Add optimal volume of solution to each well of culture plates. In case of 24 well culture plate, 0.5 ml solution is enough. Coating allows neurons to adhere to the culture plates.
    2. Leave the plates in incubator for at least 1 h. After that, wash the wells twice with 1 ml/well of sterile PBS. Completely remove the PBS before use. It is not necessary to dry the plate after PBS washing.
  2. Choose appropriate anesthesia according to the recommendations of your Institutional Animal Care and Use Committee. After anesthetization, cut the scalp and remove the skull. We use postnatal day 1 mice to measure the length of corticospinal neuron. Pick up whole brain from pups and place it in cold PBS on ice (Chen et al., 2007).
  3. Under a microscope, dissect out the cerebral cortex of brains. Remove meninges and blood vessels. The tissues are cut into as small piece (1 mm cubes) as can with sterile knives.
  4. Transfer tissues to a 50 ml tube. The tissues are trypsinized in 10 ml of the trypsin solution (0.25% trypsin, 100 μg/ml DNase in PBS), and are incubated at 37 °C for 15 min.
  5. Add an equal amount of 10% FBS-DMEM, and pipettes up and down a few times to help with tissues dissociation.
  6. Centrifuge at 300 x g for 3 min at room temperature, discard the supernatant, and resuspend the pellet in fresh DMEM (not 10% FBS-DMEM).
  7. The cells are filtered through a 70 μm nylon cell strainer to obtain single cell suspension. Centrifuge the resulting cell suspension at 300 x g for 3 min at room temperature. Resuspend the cell pellet in fresh DMEM (not 10% FBS-DMEM).
  8. Count the number of cells and adjust the concentration using 10% FBS-DMEM. To measure the neurite length, plate the cells on a 4-well chamber slide at a density of 5 x 104 cells/well. Transfer the cell suspension to culture plates and incubate for 24 h at 37 °C with 5% CO2. To investigate the effect of pharmacological agents, change to the medium containing each agent 2 h after cell plating. Representative image shows the neuronal class III β-Tubulin (Tuj-1) labeled cortical neuron obtained 1 day culture.


    Figure 1. Image of cultured cortical neuron. Representative image shows the neuronal class III β-Tubulin (Tuj-1) labeled cortical neuron obtained 1 day culture.

Recipes

  1. Poly-L-lysine: Dissolve of sterile distilled water to make stock solution (10 mg/ml). Keep aliquots at -20 °C. Dilute with sterile PBS right before use.
  2. Serum-containg culture medium
    Reagents
    Volume     
    DMEM
    445 ml
    FBS
    50 ml
    Penicillin/streptomycin
    5 ml
  3. PBS
    Reagents
    Volume
    NaH2PO4·2H2O
    0.312 g
    Na2HPO4·12H2O
    2.8652 g
    NaCl
    8.5 g
    DW
    1 L

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists (A) (25710006) from the Japan Society for the Promotion of Sciences to RM, the Osaka University Program for the Support of Networking among Present and Future Researchers to RM, and a Grant-in-Aid for Scientific Research (S) from JSPS (25221309) to TY.

References

  1. Chen, Y., Balasubramaniyan, V., Peng, J., Hurlock, E. C., Tallquist, M., Li, J. and Lu, Q. R. (2007). Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protocols 2(5): 1044-1051. 
  2. Muramatsu, R., Takahashi, C., Miyake, S., Fujimura, H., Mochizuki, H. and Yamashita, T. (2012). Angiogenesis induced by CNS inflammation promotes neuronal remodeling through vessel-derived prostacyclin. Nat Med 18(11): 1658-1664.

材料和试剂

  1. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P2636)
  2. Dulbecco's Modified Eagle Medium,DMEM(Life Technologies,Invitrogen TM ,目录号:12800-017)
  3. 胎牛血清(Life Technologies,Gibco ,目录号:10437)
  4. 2.5%胰蛋白酶溶液(Life Technologies,Gibco ,目录号:15090-046)
  5. DNase(Sigma-Aldrich,目录号:DN25)
  6. 青霉素/链霉素(Life Technologies,Gibco ,目录号:15140)
  7. NaH 2 2 PO 4 4 2H 2
  8. 2 2
  9. NaCl
  10. DMEM
  11. FBS
  12. 含血清培养基(见配方)
  13. PBS(请参阅配方)

设备

  1. 细胞培养孵化器
  2. 离心机
  3. 立体显微镜
  4. 剪刀,钳子和刀子
  5. 无菌过滤器(0.45μm)
  6. 细胞过滤器70μm尼龙(BD Biosciences,目录号:352350)
  7. 24孔培养板

程序

  1. 包衣和洗涤培养板。
    1. 用无菌PBS稀释聚-L-赖氨酸储备液至100μg/ml。向培养板的每个孔中加入最佳体积的溶液。在24孔培养板的情况下,0.5ml溶液就足够了。涂层允许神经元粘附到培养板
    2. 离开板在孵化器至少1小时。之后,用1ml /孔的无菌PBS洗孔两次。使用前完全取出PBS。在PBS洗涤之后不必干燥板。
  2. 根据您的机构动物护理和使用委员会的建议选择适当的麻醉。麻醉后,切开头皮,取出头骨。我们使用出生后第1天小鼠来测量皮质脊髓神经元的长度。从幼崽拾取整个大脑,并将其置于冰上的冷PBS中(Chen等人,2007)
  3. 在显微镜下,解剖大脑大脑皮质。去除脑膜和血管。将组织切成小块(1mm立方体),用无菌刀可以
  4. 转移组织到50ml管。将组织在10ml胰蛋白酶溶液(0.25%胰蛋白酶,100μg/ml PBS中的DNase)中胰蛋白酶化,并在37℃下孵育15分钟。
  5. 加入等量的10%FBS-DMEM,并向上和向下移液器几次以帮助组织解离。
  6. 在室温下以300×g离心3分钟,弃去上清液,并将沉淀物重新悬浮在新鲜DMEM(不是10%FBS-DMEM)中。
  7. 将细胞通过70μm尼龙细胞过滤器过滤以获得单细胞悬浮液。在室温下将所得细胞悬浮液以300×g离心3分钟。重悬细胞沉淀在新鲜的DMEM(不是10%FBS-DMEM)。
  8. 计数细胞数量,并使用10%FBS-DMEM调整浓度。为了测量神经突长度,将细胞以5×10 4个细胞/孔的密度平板在4孔腔室载玻片上。将细胞悬液转移到培养板中,并在37℃下用5%CO 2孵育24小时。为了研究药理学药剂的作用,在细胞接种后2小时改变含有每种药剂的培养基。代表图像显示获得1天培养的神经元III型β-微管蛋白(Tuj-1)标记的皮层神经元。


    图1.培养的皮层神经元的图像。代表性图像显示获得1天培养物的神经元III类β-微管蛋白(Tuj-1)标记的皮质神经元。

食谱

  1. 聚-L-赖氨酸:将无菌蒸馏水溶解以制备储备溶液(10mg/ml)。 保持等分在-20°C。 在使用前用无菌PBS稀释。
  2. 含血清培养基
    试剂
    卷      
    DMEM
    445毫升
    FBS
    50 ml
    青霉素/链霉素
    5 ml
  3. PBS
    试剂

    NaH 2 PO 4 sub> O
    0.312克
    12 = sub> O
    2.8652 g
    NaCl
    8.5克
    DW
    1 L

致谢

这项工作是由日本促进科学协会授予的青年科学家(25710006)的支持,大阪大学大学计划支持当前和未来的研究人员到RM的网络,从JSPS(25221309)到TY的科学研究授权(S)。

参考文献

  1. Chen,Y.,Balasubramaniyan,V.,Peng,J.,Hurlock,EC,Tallquist,M.,Li,J.and Lu,QR(2007)。由CNS炎症诱导的血管生成促进通过血管衍生的前列环素的神经元重塑。 Nat Med 18(11):1658-1664。
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How to cite this protocol: Muramatsu, R. and Yamashita, T. (2013). Primary Culture of Cortical Neurons. Bio-protocol 3(8): e496. DOI: 10.21769/BioProtoc.496; Full Text



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4/28/2016 9:30:01 AM  

JG4321 JG1234
HMS

In the Procedure Line 4, is 100g/ml of DNase the correct concentration?

4/29/2016 11:46:57 PM  

Rieko Muramatsu (Author)
Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Japan

100 μg/ml is correct.

5/3/2016 3:04:45 AM  

Bio-protocol Editorial Team
bio-protocol.org

We sincerely apologize for the mistake. It has been corrected accordingly in the text.

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