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[Bio101] Neuron-enriched Cultures (Method 2)
[Bio101] 神经元富集培养(方法2)   

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Abstract

Neuron-enriched cultures are a useful tool to study neuronal development and the molecular pathways that come in to play during neuronal death in various neurological disorders. This protocol is for generating midbrain neuronal cultures from late embryo rodent brain. This method uses Neurobasal medium and B27 serum-free supplement. The long-lasting (> 4 weeks) midbrain neuron-enriched cultures generated following this protocol have been wildly used to study the pathogenesis of Parkinson’s disease, the most common neurodegenerative movement disorder. The neuron-enriched cultures prepared following this protocol will contain < 10% astroglia. The usage of B27 serum-free supplement will definitively increase the cost. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

Materials and Reagents

  1. Poly-D-lysine (Sigma-Aldrich, catalog number: P7280 )
  2. DMEM/F12 (Life Technologies, Gibco®, catalog number: 11330-032 )
  3. D-Glucose (please add catalog number)
  4. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
  5. 200 mM L-glutamine (Life Technologies, Gibco®, catalog number: 25030-081 ) (100 ml)
  6. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P0781 ) (100 ml)
  7. Neurobasal medium (Life Technologies, InvitrogenTM, catalog number: 12348-017 )
  8. 50x B27 serum-free supplement (Life Technologies InvitrogenTM/Gibco®, catalog number: 21103-049 ) (10 ml)
  9. Sterile PBS
  10. MEM
  11. Trypan blue dye
  12. Poly-D-lysine solution (see Recipes)
  13. Serum-containing culture medium (see Recipes)

Equipment

  1. Cell culture incubator
  2. Centrifuge
  3. Dissection microscope
  4. Scissors and forceps
  5. 24-well plates
  6. Sterile filter (0.2 µm)
  7. Foil
  8. Laminar hood
  9. 50-ml tube
  10. 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-1. Add 0.25 ml to each well of 24-well plates. Leave the plates in the hood for 2-3 h or in the in incubator for at least 1 h.
    2. Before use, remove the coating solution. Wash the wells twice with 1 ml/well of sterile water. 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 for midbrain neuron-enriched cultures or at embryonic day 17/18 for cortical neuron-enriched cultures 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 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 serum-containing 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 serum-containing culture medium. Add 0.5 ml of cells to each well of the poly-D-lysine-coated 24-well plate. Place the plates in a humidified 37 °C incubator with 5% CO2.
  9. Two days later, change cultures to 1 ml/well serum-free neurobasal medium.
  10. Treat seven-day-old cultures that contain less than 0.1% OX-42-immunoreactive microglia and about 3% 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. Serum-containing culture medium
    Reagents
    Volume
    Final con.
    DMEM/F12
    445 ml
    -
    D-Glucose
    3.0 g
    6 g/L
    Heat-inactivated fetal bovine serum*
    50 ml
    10%
    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.
  3. Serum-free neurobasal medium
    Reagents
    Volume Final con.
    Neurobasal medium
    483.75 ml
    -
    B27 serum-free supplement
    10 ml
    1x
    L-glutamine
    1.25 ml
    0.5 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.
  2. Lotharius, J., Dugan, L. L. and O'Malley, K. L. (1999). Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons. J Neurosci 19(4): 1284-1293.

简介

神经元富集的培养物是研究神经元发育和在神经元死亡期间在各种神经疾病中发挥的分子途径的有用工具。 该协议是用于从晚期胚胎啮齿动物脑产生中脑神经元培养物。 该方法使用Neurobasal培养基和B27无血清补充剂。 根据该方案产生的持续(> 4周)中脑神经元富集培养物已被广泛用于研究帕金森病的发病机理,帕金森病是最常见的神经退行性运动障碍。 按照该方案制备的富含神经元的培养物将含有<10%星形胶质细胞。 使用B27无血清补充剂将明显增加成本。 这个协议已经由Hong博士实验室的各种研究人员,尤其是刘博士多年来开发和改进。

材料和试剂

  1. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P7280)
  2. DMEM/F12(Life Technologies,Gibco ,目录号:11330-032)
  3. D-葡萄糖(请添加目录号)
  4. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000-044)
  5. 200mM L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)(100ml)
  6. 青霉素/链霉素(Sigma-Aldrich,目录号:P0781)(100ml)
  7. Neurobasal培养基(Life Technologies,Invitrogen TM ,目录号:12348-017)
  8. 50×B27无血清补充物(Life Technologies Invitrogen /Gibco ,目录号:21103-049)(10ml)
  9. 无菌PBS
  10. MEM
  11. 台盼蓝染料
  12. 聚-D-赖氨酸溶液(参见配方)
  13. 含血清培养基(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. 24孔板
  6. 无菌过滤器(0.2μm)

  7. 层流罩
  8. 50 ml管
  9. 10 ml移液器

程序

  1. 涂覆和清洗培养皿
    1. 在层流罩中,用无菌稀释聚D-赖氨酸储备溶液(5x)   水至20μg/ml。 向24孔板的每个孔中加入0.25ml。 离开 在罩中的板2-3小时或在孵化器至少1 H。
    2. 使用前,取下涂层溶液。 洗涤孔两次   用1ml /孔的无菌水。 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,从时间怀孕的大鼠或小鼠胚胎的第13/14期为中脑神经元富集培养物或在胚胎17/18的胚胎的富含皮质神经元的培养物中移除胚胎,并将胚胎置于冷的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毫升吸管,然后1毫升移液管吸头安装到10毫升吸管,随后用一个适合的200微升吸头。
  5. 以6.5倍速度设定(〜1,500rpm)将研磨的组织离心10分钟
  6. 小心地除去上清液,并将沉淀的细胞重悬在10ml含血清的培养基中
  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计数细胞密度。
  8. 用含血清的培养基调节细胞密度至1×10 6个细胞/ml。 向聚-D-赖氨酸包被的24孔板的每个孔中加入0.5ml细胞。 将板放置在具有5%CO 2的湿润的37℃培养箱中
  9. 两天后,将培养物更换为1ml /孔无血清的neurobasal培养基
  10. 用期望的试剂或载体处理含有小于0.1%OX-42-免疫反应性小胶质细胞和约3%神经胶质纤维酸性蛋白(GFAP) - 免疫反应性星形胶质细胞的7天龄培养物。

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. 含血清培养基
    试剂

    最终结果。
    DMEM/F12
    445毫升
    -
    D-葡萄糖
    3.0克
    6 g/L
    热灭活的胎牛血清*
    50 ml
    10%
    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。
  3. 无血清neurobasal培养基
    试剂
    最终结果。
    神经基质培养基
    483.75 ml
    -
    B27无血清补液剂
    10 ml
    1x
    L-谷氨酰胺 1.25 ml
    0.5 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。
  2. Lotharius,J.,Dugan,L.L。和O'Malley,K.L。(1999)。 独特的机制是培养的多巴胺能神经元中神经毒素介导的细胞死亡的基础。 Neurosci 19(4):1284-1293
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gao, H. (2011). Neuron-enriched Cultures (Method 2). Bio-protocol Bio101: e150. DOI: 10.21769/BioProtoc.150;
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