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This protocol will guide you through the process for generating midbrain neuronal cultures from late embryo mouse brain. These cultures serve as a useful tool to study molecular pathways during neuronal death in various neurological disorders. This method uses cytosine β-D-arabinofuranoside in the cultures to suppress the proliferation of glial cells. Seven days after the seeding, the neuron-enriched cultures prepared following this protocol will contain less than 10% glia cultures. The concentrations of β-D-arabinofuranoside should be adjusted according to your culture condition. 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 1)
[Bio101] 神经元富集培养(方法1)

神经科学 > 细胞机理 > 细胞分离和培养
作者: 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, 6049 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.151

[Abstract] This protocol will guide you through the process for generating midbrain neuronal cultures from late embryo mouse brain. These cultures serve as a useful tool to study molecular pathways during neuronal death in various neurological disorders. This method uses cytosine β-D-arabinofuranoside in the cultures to suppress the proliferation of glial cells. Seven days after the seeding, the neuron-enriched cultures prepared following this protocol will contain less than 10% glia cultures. The concentrations of β-D-arabinofuranoside should be adjusted according to your culture condition. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

[Abstract] 本方法将引导您完成从后期胚胎小鼠大脑产生中脑神经元培养的整个过程。这种培养是来研究在神经元死亡中各种神经系统疾病的分子通路的有用工具。这种方法使用胞嘧啶β-D-arabinofuranoside(阿糖胞嘧啶)到培养基中,抑制胶质细胞增生。播种后第7天,在本方法后的神经元富集的准备将含有不少于10%的神经胶质细胞培养基。β-D-arabinofuranoside的浓度应根据培养基条件调整。此实验方法经Dr. Hong实验室不同研究人员多年的发展和完善,尤其是Dr. Bin Liu.

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 PBS
  5. Trypan blue dye
  6. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
  7. Heat-inactivated horse serum (HS) (Life Technologies, Gibco®, catalog number: 26050-088 )
  8. Non-essential amino acids (100 ml) (Life Technologies, Gibco®, catalog number: 11140-050 )
  9. Sodium pyruvate (100 ml) (Sigma-Aldrich, catalog number: S8636 )
  10. 200 mM L-glutamine (100 ml) (Life Technologies, Gibco®, catalog number: 25030-081 )
  11. Penicillin/streptomycin (100 ml) (Sigma-Aldrich, catalog number: P0781 )
  12. Neurobasal medium (Life Technologies, InvitrogenTM, catalog number: 12348-017 )
  13. 50x B27 serum-free supplement (10 ml) (Life Technologies, InvitrogenTM/ Gibco®, catalog number: 21103-049 )
  14. Poly-D-lysine solution (see Recipes)
  15. Maintenance culture medium (see Recipes)
  16. Treatment medium (see Recipes)

Equipment

  1. Cell culture incubator
  2. Centrifuges
  3. Dissection microscope
  4. Scissors and forceps
  5. Laminar hood
  6. 24-well plates
  7. 50 ml tube
  8. 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 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 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 to 1 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. Two days after initial seeding, add 0.5 ml culture medium containing 10–20 µM cytosine β-D-arabinofuranoside to the cultures to suppress the proliferation of glial cells.
  12. At 2–3 d later, change the cultures back to fresh culture medium.
  13. Treat seven-day-old cultures that contain less than 0.1% OX-42-immunoreactive microglia and about 8% 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. 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.

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.

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PBS
  5. 台盼蓝染料
  6. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000-044)
  7. 热灭活的马血清(HS)(Life Technologies,Gibco ,目录号:26050-088)
  8. 非必需氨基酸(100ml)(Life Technologies,Gibco ,目录号:11140-050)
  9. 丙酮酸钠(100ml)(Sigma-Aldrich,目录号:S8636)
  10. 200mM L-谷氨酰胺(100ml)(Life Technologies,Gibco ,目录号:25030-081)
  11. 青霉素/链霉素(100ml)(Sigma-Aldrich,目录号:P0781)
  12. Neurobasal培养基(Life Technologies,Invitrogen TM ,目录号:12348-017)
  13. 50×B27无血清补充物(10ml)(Life Technologies,Invitrogen /Gibco ,目录号:21103-049)
  14. 聚-D-赖氨酸溶液(参见配方)
  15. 维护培养基(参见配方)
  16. 处理介质(见配方)

设备

  1. 细胞培养孵化器
  2. 离心机
  3. 解剖显微镜
  4. 剪刀和镊子
  5. 层流罩
  6. 24孔板
  7. 50ml管
  8. 10 ml移液器

程序

  1. 涂覆和清洗培养皿
    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,从怀孕的大鼠或小鼠在胚胎的第13/14天除去胚胎,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10ml移液管,然后1ml移液管吸头安装到10ml移液管,随后用一个适合的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. 在初始接种后两天,向培养物中加入0.5ml含有10-20μM胞嘧啶β-D-阿拉伯呋喃糖苷的培养基以抑制胶质细胞的增殖。
  12. 2-3 d后,将培养基更换回新鲜培养基
  13. 用含有少于0.1%OX-42-免疫反应性小胶质细胞和约8%胶质纤维酸性蛋白(GFAP) - 免疫反应性星形胶质细胞的7天龄培养物与所需试剂或载体一起处理。

食谱

  1. 聚-D-赖氨酸溶液
    溶解在50ml ddH 2 O中,制成5x储备溶液 保持5.0 ml等分试样在-20°C
  2. 维护培养基
    试剂

    最终结果。
    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°C热灭活30分钟,并以50 ml等分试样储存在-70°C。
    **以-20毫升等分试样储存于-20℃
  3. 处理介质
    试剂

    最终结果。
    MEM
    465毫升
    -
    热灭活的FBS
    10 ml
    2%
    热灭活的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

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不同作用 Neurosci 22(3):782-790。
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How to cite this protocol: Gao, H. (2011). Neuron-enriched Cultures (Method 1). Bio-protocol Bio101: e151. DOI: 10.21769/BioProtoc.151; Full Text



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