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Adult dorsal root ganglia neurons are among the few adult neuronal cell types that can be purified and grown relatively easily in dissociated cell culture. Here we describe a procedure for the isolation and growth of dissociated adult mouse DRG neurons using Percoll gradients and a chemically defined medium. These cultures can be used for morphological, biochemical and electrophysiological studies of neuronal growth and function.

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Isolation and Growth of Adult Mouse Dorsal Root Ganglia Neurons
成年小鼠背根神经节神经元的分离和生长

神经科学 > 细胞机理 > 细胞分离和培养
作者: Seong-il Lee
Seong-il LeeAffiliation: Department of Neurobiology and Behavior, Stonybrook University, Stonybrook, USA
For correspondence: silee7e@gmail.com
Bio-protocol author page: a2568
 and Joel Levine
Joel LevineAffiliation: Department of Neurobiology and Behavior, Stonybrook University, Stonybrook, USA
Bio-protocol author page: a2569
Vol 5, Iss 18, 9/20/2015, 5964 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1601

[Abstract] Adult dorsal root ganglia neurons are among the few adult neuronal cell types that can be purified and grown relatively easily in dissociated cell culture. Here we describe a procedure for the isolation and growth of dissociated adult mouse DRG neurons using Percoll gradients and a chemically defined medium. These cultures can be used for morphological, biochemical and electrophysiological studies of neuronal growth and function.
Keywords: Primary culture(原代培养), Dorsal root ganglia(背根节), Neuron(神经元), Percoll gradient(Percoll梯度), Rodent(rodent)

[Abstract]

Materials and Reagents

  1. 6 to 9 weeks old female mouse
  2. Poly-L-lysine hydrobromide (100 μg/ml in water) (Sigma-Aldrich, catalog number: P1524 )
  3. Laminin (1 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 23017-015 )
  4. Collagenase from Clostridium histolyticum (10 mg/ml in Ca2+, Mg2+ free PBS) (Sigma-Aldrich, catalog number: C-9891 )
  5. Dispase II (10 mg/ml in Ca2+, Mg2+ free PBS) (Roche Diagnostics, catalog number: 04-942-078-001 )
  6. Deoxyribonuclease I from bovine pancreas (4 mg/ml in PBS) (Sigma-Aldrich, catalog number: DN25 )
  7. Leibovitz's L-15 Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11415-064 )
  8. Neurobasal®-A Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888-022 )
  9. B-27® supplement (50X), serum free (Thermo Fisher Scientific, InvitrogenTM, catalog number: 17504-044 )
  10. Percoll® (Sigma-Aldrich, catalog number: P1644 )
  11. Hank’s Balanced Salt Solution (HBSS, calcium, magnesium, no phenol red) (Thermo Fisher Scientific, GibcoTM, catalog number: 1425092 )
  12. 15 mm Glass coverslips (Neuvitro, catalog number: GG-15 )
  13. Pasteur pipette
  14. Falcon® 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish, 20/Pack, 500/Case, Sterile (Corning Incorporated, Falcon®, catalog number: 351008 )
  15. Falcon® 60 mm x 15 mm Not TC-Treated Treated Bacteriological Petri Dish, 20/Pack, 500/Case (Corning Incorporated, Falcon®, catalog number: 351007 )
  16. Falcon® 14 ml Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap, Sterile, 25/Pack, 500/Case (Corning Incorporated, Falcon®, catalog number: 352059 )
  17. Falcon® 14 ml Round Bottom PP Test Tube, without Cap, Sterile, 125/Pack, 1000/Case (Corning Incorporated, Falcon®, catalog number: 352018 )

Equipment

  1. #10 scalpel blade (Fine Science Tool, catalog number: 10010-00 ) (Figure 1)
  2. Standard scalpel handle #3 (Fine Science Tool, catalog number: 10003-12 ) (Figure 1)
  3. Spring Scissors-Angled to Side (Fine Science Tool, catalog number: 15006-09 ) (Figure 1)
  4. Small curved scissors (Fine Science Tool, catalog number: 14095-11 ) (Figure 1)
  5. Standard Pattern Forceps (Fine Science Tool, catalog number: 11000-12 ) (Figure 1)
  6. Dumont #5 forceps (Fine Science Tool, catalog number: 11252-20 ) (Figure 1)
  7. Dissecting microscope
  8. Swinging bucket rotor (Sorvall instrument, model: HB-4 )
  9. Centrifuge (Sorvall instrument, model: RC5 plus )


    Figure 1. The dissection tools for mouse dissection and DRG isolation. A. Standard scalpel handle #3; B. Spring scissors; C. Small curved scissors; D. Standard forceps; E. Dumont #5 forceps.

Procedure

  1. Preparation of coverslips for DRG neuronal culture
    1. The day before the dissection, place 1 sterilized glass coverslip into a 35 mm Petri-dish. Cover with 250 μl of poly-L-lysine (PLL) (100 μg/ml in water) and incubate at 37 °C overnight. (Tissue culture wells can also be used here.)
    2. The day of dissection, aspirate off the PLL solution from each coverslip and add 250 μl of sterile phosphate-buffered saline (PBS) on the coverslip. Repeat this 3 times. After third wash, leave the PBS solution on the coverslip until the laminin solution is ready at room temperature.
    3. Thaw laminin aliquot slowly on ice and dilute it to 2~5 μg/ml in PBS. Remove PBS from the coverslip and add 200 μl of the diluted laminin onto the PLL-coated coverslip.
    4. Incubate the coverslips at 37 °C at least 2 h before plating cells.
      Note: Other proteins of interest, such as BSA, chondroitin sulfate proteoglycans, Nogo and myelin extract can be added to the laminin solution. Laminin is required for extensive growth of DRG neurites.

  2. Dissection and isolation of the dorsal root ganglion
    1. Euthanize a 6 to 9 weeks old female mouse via CO2 inhalation. Shave the back and wash the skin with 70% ethanol (Figure 2A).


      Figure 2. Mouse dissection. A. Shave the back and wash the skin with 70% ethanol; B. Make a midline incision along the back with a scalpel; C. Cut through the lumbar end of the spine; D. Cut the cervical end of the spinal cord and remove to a 60 mm petri dish.

    2. Make a midline incision along the back with a scalpel and deflect the skin laterally (Figure 2B). Using a small curved scissors cut through the lumbar end of the spine (Figure 2C). Hold and lift up the incised lumbar part with a forceps and cut through the ribs and bones along both sides. Cut off any attached connective tissues attached to the sides and underneath the spine (Figure 2D). Using a small sharp scissors, cut the cervical end of the spinal cord and remove to a 60 mm petri dish. Carefully remove the ribs, muscle and connective tissues as much as possible. Cut the cleaned spinal column into three pieces (Figure 3A and B).
    3. Put one blade of spring scissors between dorsal side of vertebral column and spinal canal and carefully cut through vertebral column. Repeat this along the ventral side and pull apart the right and left halves of vertebral column (Figure 3C and D).
    4. Place the spine pieces into L15 media at room temperature (RT). Remove the spinal cord using a fine forceps (#5 Dumont). Under a dissecting microscope, locate the DRGs which sit in small cavities along the lateral vertebral column. Place the tips of a #5 forceps underneath the DRG, pluck it out and transfer it to the petri-dish containing L15 media.
    5. Using fine forceps, remove any fibrous structures surrounding the DRGs and cut off any attached nerve roots. Transfer the DRGs into 15 ml tube filled with L15 media.


      Figure 3. Spine dissection. A. Remove the ribs, muscle and connective tissues from a spine column; B. Cut the cleaned spinal column into three pieces; C. Cut through the vertebral column from dorsal to ventral side; D. Pull apart the right and left halves of vertebral column.

    6. Centrifuge it at 200 x g for 2 min. Remove the media and replace with 1 ml of L15 media. Add 200 μl each of collagenase and Dispase II solution. Gently mix the suspension and incubate at 37 °C for 20 min.
    7. Remove the enzyme solution carefully and add 2 ml of L15.
    8. Triturate the solution with fire-polished glass Pasteur pipette, add 25 μl of DNaseI and incubate the cell suspension at 37 °C for 20 min.
    9. Repeat the trituration again and add 5 ml of L15.
    10. Spin down the cells at 200 x g for 5 min and remove the supernatant and resuspend the cell pellet in 5 ml of L15.
    11. Repeat this step one more time and resuspend the cell pellet in 2 ml of L15 media.


      Figure 4. Isolation of DRGs. A. Dissected vertebral column, Arrow and Arrow head; spinal cord; B. Vertebral column after spinal cord was removed. Arrowheads; DRGs sitting in the spine; C. Arrowheads; Isolated DRGs, Open Arrow heads: DRG fibers.

  3. Purification of DRG neurons by using Percoll gradients
    1. Add 1 ml of 10x Hank’s Balanced Salt Solution (HBSS) to 9 ml of Percoll and mix thoroughly to make a 90% working solution.
    2. Dilute 90% Percoll in 1x HBSS to make stock solutions of 30 and 60% Percoll.
    3. Place 3 ml of 60% Percoll in 14 ml round bottom tube and carefully overlay with 3 ml of 30% Percoll. It is important not to disturb the interface between the 2 concentrations of Percoll.
    4. Place 1 ml of the cell suspension over the Percoll gradient solution being careful to not disturb the interface of Percoll.
    5. Centrifuge the tubes at 800 x g for 20 min at 4 °C using a swinging bucket rotor.
    6. After centrifugation, the cloudy layer at the interface of the two Percoll solutions contains the DRG neurons (Figures 5 and 6). Gently remove and discard the first 3 ml from the top which contains mainly myelin and Schwann cell debris. Transfer the next 2 ml containing the neurons into 15 ml conical tube.
    7. Fill up the 15 ml tube with L15 media. Centrifuge it at 200 x g for 10 min.
    8. Remove the supernatant and suspend the cell pellet in 1 ml of Neurobasal-A supplemented with B-27.
    9. Count cell number. We usually obtain about 10,000 viable DRG neurons from a single mouse which is sufficient for 8-10 15 mm round coverslips with a proper density for morphological studies such as neurite length, polarization, etc.


      Figure 5. An example of Percoll gradient. Arrowhead; Schwann cell layer; Arrow; DRGs layer.


      Figure 6. Isolation of neuronal cell by Percoll gradient. A. The microscopic image of cell suspension before Percoll gradient; B. The microscopic image of cell suspension acquired from top layer after Percoll gradient; C. The microscopic image of cell suspension acquired from middle layer after Percoll gradient. Arrow; DRG neurons; Arrow head; Schwann cells.

    10. Wash the laminin-coated coverslips with PBS three times. After aspirating off the third wash, immediately plate 150 μl of cell suspension onto each coverslip. Incubate the cultures at 37 °C for 1 h and then feed an additional 1 ml of Neurobasal-A supplemented with B-27 into each 35 mm petri-dish containing the coverslip.
    11. After about 24 h, extensive neurite outgrowth should be observed (Figure 7). Keep monitoring the growth. For neurite growth experiments, we usually fix the culture 48 h after plating.


      Figure 7. A representative picture of DRG neurons in vitro culture. Dissociated DRG neurons from adult mice were grown on coverslips coated with 2 µg/ml of laminin. After 48 h, the cultures were fixed and immunostained with anti-TUJ antibody.

Acknowledgments

This work was supported by a grant from Spinal Cord Injury research board of New York State Department of Health.

References

  1. DeBoni, U. and Goldberg, S. S. S. (1989). Isolation of pure fractions of viable dorsal rot ganglionic neurons from rabbit or mouse using Percoll Gradients. In: Shahar, deVellis, Vernadakis and Haber (eds). A dissection and tissue culture manual of the nervous system. Alan R Liss, Inc., 230-232.
  2. Lee, S. I., Zhang, W., Ravi, M., Weschenfelder, M., Bastmeyer, M. and Levine, J. M. (2013). Atypical protein kinase C and Par3 are required for proteoglycan-induced axon growth inhibition. J Neurosci 33(6): 2541-2554.

材料和试剂

  1. 6至9周龄的雌性小鼠
  2. 聚-L-赖氨酸氢溴酸盐(100μg/ml,在水中)(Sigma-Aldrich,目录号:P1524)
  3. 层粘连蛋白(1mg/ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:23017-015)
  4. 来自溶组织梭菌的胶原酶(10mg/ml Ca 2+ + Mg 2+游离PBS)(Sigma-Aldrich,目录号:C-9891)
  5. 分散酶II(10mg/ml,在Ca 2+ + Mg 2+游离PBS中)(Roche Diagnostics,目录号:04-942-078-001)
  6. 来自牛胰腺的脱氧核糖核酸酶I(PBS中4mg/ml)(Sigma-Aldrich,目录号:DN25)
  7. Leibovitz's L-15 Medium(Thermo Fisher Scientific,Gibco TM ,目录号:11415-064)
  8. Neurobasal -A Medium(Thermo Fisher Scientific,Gibco TM ,目录号:10888-022)
  9. 无血清(Thermo Fisher Scientific,Invitrogen TM ,目录号:17504-044)的B-27补充(50X)
  10. Percoll(Sigma-Aldrich,目录号:P1644)
  11. Hank's平衡盐溶液(HBSS,钙,镁,无酚红)(Thermo Fisher Scientific,Gibco TM ,目录号:1425092)
  12. 15mm玻璃盖玻片(Neuvitro,目录号:GG-15)
  13. 巴斯德吸管
  14. Falcon 35mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish,20/Pack,500/Case,Sterile(Corning Incorporated,Falcon ,目录号:351008 )
  15. 60/15mm未处理的TC处理的细菌培养皿,20 /包,500 /箱(Corning Incorporated,Falcon ,目录号:351007)
  16. Falcon ? 14 ml圆底高清PP试管, 刻度,带扣帽,无菌,25 /包,500 /箱(Corning Incorporated,Falcon ,目录号:352059)
  17. 14L圆底PP试管,无盖,无菌,125 /包,1000 /箱(Corning Incorporated,Falcon ,目录号:352018)

设备

  1. #10手术刀刀片(Fine Science Tool,目录号:10010-00)(图1)
  2. 标准手术刀柄#3(Fine Science Tool,目录号:10003-12)(图1)
  3. Spring Scissors-Angled to Side(Fine Science Tool,目录号:15006-09)(图1)
  4. 小型剪刀(Fine Science Tool,目录号:14095-11)(图1)
  5. 标准模式钳(Fine Science Tool,目录号:11000-12)(图1)
  6. Dumont#5镊子(Fine Science Tool,目录号:11252-20)(图1)
  7. 解剖显微镜
  8. 摆动转子(Sorvall仪器,型号:HB-4)
  9. 离心机(Sorvall仪器,型号:RC5 plus)


    图1.用于小鼠解剖和DRG隔离的解剖工具。 A.标准解剖刀柄#3; B.弹簧剪刀; C.小弯曲剪刀;标准镊子; E. Dumont#5镊子

程序

  1. 制备DRG神经元培养的盖玻片
    1. 在解剖前一天,将1块无菌玻璃盖玻片放入 ?35mm培养皿。盖上250微升的聚-L-赖氨酸(PLL)(100微克/毫升 在水中)并在37℃孵育过夜。 (组织培养孔可以 也在这里使用。)
    2. 解剖的日子,吸出PLL 溶液从每个盖玻片和添加250微升无菌 磷酸盐缓冲盐水(PBS)在盖玻片上。重复3次。 第三次洗涤后,离开PBS溶液在盖玻片上,直到 层粘连蛋白溶液在室温下就绪
    3. 解冻层粘连蛋白 缓慢等分在冰上,并在PBS中稀释至2?5μg/ml。从中移除PBS ?盖玻片并加入200μl稀释的层粘连蛋白到PLL包被的 ?盖玻片
    4. 在37℃孵育盖玻片至少2小时,然后铺板细胞 注意:其他感兴趣的蛋白质,如BSA,硫酸软骨素 蛋白聚糖,Nogo和髓鞘提取物可以添加到层粘连蛋白中 解。层粘连蛋白是DRG神经突的大量生长所需的。

  2. 背根神经节的解剖和隔离
    1. 通过CO 2吸入安乐死6至9周龄的雌性小鼠。刮去背部,用70%乙醇洗涤皮肤(图2A)

      图2.小鼠解剖。A.剃刮背部并用皮肤清洗 70%乙醇; B.用手术刀沿背部切开中线切口; C.穿过脊柱的腰部;切开子宫颈端 脊髓,并取出到60毫米培养皿
    2. 做一个 中线切口沿着背部用手术刀并偏转皮肤 (图2B)。使用一把小弯曲的剪刀剪 腰脊柱(图2C)。举起并举起切开的腰部 ?部分用镊子和切开通过肋骨和骨头沿二 侧面。切除附着在侧面的任何附着的结缔组织 ?在脊柱下(图2D)。使用小锋利的剪刀,切 ?颈部末端,并取出至60mm培养皿。 小心地去除肋骨,肌肉和结缔组织 可能。将清洁的脊柱切成三片(图3A和图3B) ?B)。
    3. 在背面之间放一片弹簧剪刀 脊柱和椎管,并仔细切穿椎体 柱。沿着腹侧重复这个过程,并向右和向左拉 脊柱左半部(图3C和D)
    4. 放置 脊柱片在室温(RT)下进入L15培养基。去除脊柱 使用细镊子(#5 Dumont)。在解剖显微镜下, 找到位于沿着外侧椎骨的小腔中的DRG 柱。将#5镊子的提示放在DRG下面,拔出 并将其转移到含有L15培养基的培养皿中。
    5. 使用 细镊子,去除围绕DRGs和切割的任何纤维结构 ?关闭任何附加的神经根。转移DRGs到15毫升管填充 与L15媒体。


      图3.脊柱剥离 A.去除肋骨, 来自脊柱的肌肉和结缔组织; B.切割清洁 脊柱三段; C.穿过脊柱 从背侧到腹侧; D.拉开右半边和左半边 脊柱。

    6. 以200×g离心2分钟。去掉 ?培养基并用1ml L15培养基替换。加入200μl 胶原酶和Dispase II溶液。轻轻混合悬浮液 在37℃孵育20分钟
    7. 小心地除去酶溶液,加入2ml L15
    8. 用火抛光玻璃巴斯德吸管研磨溶液,加入 ?25μlDNaseI,并将细胞悬浮液在37℃下孵育20分钟
    9. 再次重复研磨,加入5ml L15
    10. 在200×g下旋转细胞5分钟,除去上清液,并将细胞沉淀重悬在5ml的L15中。
    11. 重复此步骤一次,并将细胞沉淀重悬在2ml L15培养基中

      图4. DRG的隔离。A.解剖的脊柱,箭头和 箭头;脊髓; B.脊柱后的脊柱 删除。箭头;坐在脊椎上的DRG; C.箭头;孤立 DRG,开口箭头:DRG光纤。

  3. 使用Percoll梯度纯化DRG神经元
    1. 将1ml 10x Hank's平衡盐溶液(HBSS)加入9ml Percoll中,充分混合,制成90%工作溶液。
    2. 在1×HBSS中稀释90%Percoll以制备30和60%Percoll的储备溶液
    3. 放置3毫升60%Percoll在14毫升圆底管,小心 用3ml的30%Percoll覆盖。重要的是不要打扰 界面之间的2个浓度的Percoll。
    4. 将1ml的细胞悬液在Percoll梯度溶液中,小心不要打扰Percoll的界面
    5. 使用摆动转子在4℃下以800×g离心试管20分钟。
    6. 离心后,在两者的界面处有浑浊层 Percoll溶液含有DRG神经元(图5和6)。轻轻地 从顶部删除和丢弃第一个3毫升主要包含 髓鞘和施万细胞碎片。转移下2毫升含有 神经元进入15ml锥形管。
    7. 填充15毫升管与L15媒体。以200×g离心10分钟。
    8. 除去上清液并将细胞沉淀悬浮在1ml补充有B-27的Neurobasal-A中。
    9. 计数单元格编号。我们通常获得大约10,000个可行的DRG神经元 ?从一只鼠标,这是足够的8-10 15毫米圆盖玻片 ?具有用于形态学研究的适当密度,例如神经突长度, ?偏振,等。


      图5. Percoll渐变的示例:箭头;施万细胞层;箭头; DRGs层。


      图6.通过Percoll梯度分离神经元细胞。 A. 在Percoll梯度之前的细胞悬液的显微镜图像; B. The 从顶层获得的细胞悬液的显微镜图像 Percoll梯度; C.获得的细胞悬液的显微镜图像 从中层后Percoll梯度。箭头; DRG神经元;箭头 头;施万细胞。

    10. 洗涤层粘连蛋白包被的盖玻片 与PBS三次。吸出第三次洗涤后,立即 在每个盖玻片上加入150μl细胞悬浮液。孵化 在37℃培养1小时,然后再加入1ml 在每个35mm培养皿中补充B-27的Neurobasal-A 含有盖玻片。
    11. 约24小时后,大量神经突 应观察到生长(图7)。持续监控增长。对于 ?神经突生长实验,我们通常在48小时后修复培养 电镀。


      图7.DRG神经元在体外培养中的代表性图片。来自成年小鼠的解离的DRG神经元在 用2μg/ml层粘连蛋白包被的盖玻片。 48小时后,培养物 ?固定并用抗TUJ抗体免疫染色。

致谢

这项工作得到纽约州卫生部脊髓损伤研究委员会的资助。

参考文献

  1. DeBoni,U.and Goldberg,S.S.S.(1989)。使用Percoll梯度从兔或小鼠分离活的背侧神经节神经节的纯级分。在:Shahar,deVellis,Vernadakis和Haber(eds)。 神经系统的解剖和组织培养手册。 Alan R Liss,Inc.,230-232。
  2. Lee,S.I.,Zhang,W.,Ravi,M.,Weschenfelder,M.,Bastmeyer,M.and Levine,J.M。 非典型蛋白激酶C和Par3是蛋白聚糖诱导的轴突生长抑制所需的。 em> J Neurosci 33(6):2541-2554。
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How to cite this protocol: Lee, S. and Levine, J. (2015). Isolation and Growth of Adult Mouse Dorsal Root Ganglia Neurons. Bio-protocol 5(18): e1601. DOI: 10.21769/BioProtoc.1601; Full Text



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(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


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