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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, 5663 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.

材料与方法

 

1.        多聚-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.        非必需氨基酸(Gibco 11140-050; 100 ml)

7.        丙酮酸钠(Sigma; S8636; 100 ml)

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

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

10.    神经基质培养基 (Invitrogen 12348-017)

11.    B27无血清补充物(Invitrogen/Gibco 21103-049; 10ml; 50X)

 

设备

 

1.          细胞培养孵化器

2.          离心机

3.          解剖镜

4.          剪刀和镊子

 

实验步骤

 

1.          包被和清洗培养板 

1)      在层式通风橱中用灭菌水稀释多聚-D-赖氨(5X) 20 μg/ml 24孔板每孔中加入0.25 ml。将板放入通风橱 2-3 h 或在至少1 h

2)      使用前,移除包被液。每孔用1ml灭菌水清洗孔两次。每孔加入1 ml 灭菌 PBS 在使用前彻底移除PBS     

2.          在动物操作室,从怀孕期的大鼠或小鼠中移出胚胎期13/14天的胚胎供中脑神经元富集培养,并把胚胎放于冷的MEM

3.          在显微镜下,解剖取出大鼠或小鼠胚胎大脑的中脑部分。移除脑膜和血管。集中组织并保存在冰冷的MEM中。

4.          在层式通风橱中,转移组织到50-ml管中. 温和碾碎组织(每个5-10),先用10ml移液器,再用一个适合于10ml移液器的1ml 移液器枪头,接着用一个适合的200-μl 移液器枪头

5.          离心研磨组织10 min ,于6.5X速度设定 (~1500 rpm).   

6.          小心移除上清,并在10 ml细胞培养维持液混悬经压片的细胞。

7.          30 μl细胞悬液,与270 μl锥虫蓝染料混合。装载10 μl至血球计数器以计算细胞密度。

8.          细胞培养维持液调整细胞浓度至1 X 106 细胞/ml 多聚-D-赖氨包被的24孔板每孔加入 0.5 ml 细胞。将板放于增湿的 37°C 培养器中,含有5% CO2. 

9.          初始播种两天后,在培养中加入0.5 ml 培养基含有10–20 μM的胞嘧啶β-D-arabinofuranoside 以抑制神经胶质细胞增殖。

10.      2–3 天后, 更换成新鲜培养基。

11.      用所需试剂或媒介物处理培养时间7天的细胞,其中含有少于0.1% OX-42-免疫反应的小胶质细胞,以及大8%的神经胶质原纤维酸性蛋白质(GFAP) -免疫反应的星形胶质细胞

 

配方

 

1.          多聚-D-赖氨酸(Sigma P-7280):  溶解在50 ml ddH2O制作 5X储液. 5.0ml分装于-20存储使用前用无菌ddH2O稀释。

2.          细胞培养维持液

 

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%

非必需氨基酸

5 ml

0.1 mM

丙酮酸钠

5 ml     

1 mM

L-谷氨酸glutamine

5 ml

2 mM

青霉素/链霉素

5 ml

50 U/ml/50 μg/ml

 

无菌滤器(0.2 μm)过滤,并用铝箔包裹存储于4°C.

*热灭活56°C  30 min并在50ml分装于-70存储

**50ml分装-20存储.

3.          处理介质

试剂                                                           体积                     终浓度

MEM                                                           465 ml                    -

热灭活胎牛血清 FBS                                      10 ml                    2%

热灭活马血清HS                                          10 ml                    2%

丙酮酸钠                                                        5 ml                    1 mM

L-谷氨酸                                                          5 ml                   2 mM

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

无菌滤器(0.2 μm)过滤,并用铝箔包裹存储于4°C

 

参考文献

 

1.         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. (2011). Neuron-enriched Cultures (Method 1). Bio-protocol Bio101: e151. DOI: 10.21769/BioProtoc.151; Full Text



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