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Neurite outgrowth in culture provides an easy way to determine the effects of a particular substrate or exogenous factor on neuron behavior. Dissociated neurons can be plated on a variety of substrates and the length of the longest neurite outgrowth can be compared. Here, we describe how to isolate and dissociate dorsal root ganglion (DRG) neurons, culture them on coverslips, and measure longest neurite outgrowth.

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Neurite Outgrowth Assay
神经突增生试验

神经科学 > 细胞机理 > 细胞分离和培养
作者: Angela R. Filous
Angela R. FilousAffiliation: Department of Neurosciences, Case Western Reserve University, Cleveland, USA
For correspondence: arn29@case.edu
Bio-protocol author page: a2822
 and Jerry Silver
Jerry SilverAffiliation: Department of Neurosciences, Case Western Reserve University, Cleveland, USA
For correspondence: jxs10@case.edu
Bio-protocol author page: a2823
Vol 6, Iss 1, 1/5/2016, 2307 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1694

[Abstract] Neurite outgrowth in culture provides an easy way to determine the effects of a particular substrate or exogenous factor on neuron behavior. Dissociated neurons can be plated on a variety of substrates and the length of the longest neurite outgrowth can be compared. Here, we describe how to isolate and dissociate dorsal root ganglion (DRG) neurons, culture them on coverslips, and measure longest neurite outgrowth.
Keywords: Neurite outgrowth(神经突起生长), Dorsal root ganglion (DRG)(背根神经节(DRG)), Glia(神经胶质细胞), Laminin(层粘连蛋白)

[Abstract]

Materials and Reagents

  1. 12 mm glass coverslips
  2. 24 well plate
  3. Pipette tips
  4. Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P1274 )
  5. Laminin Mouse Protein, Natural (Invitrogen, catalog number: 23017-015 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 23017-015”.
  6. HBSS-CMF (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14185-052 )
  7. Collagenase II (Worthington Biochemical Corporation, catalog number: 4176 )
  8. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
  9. Penicillin-streptomycin (Invitrogen, catalog number: 15140122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140122”.
  10. B-27® Serum-Free Supplement (50x) (Invitrogen, catalog number: 17504-044 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044”.
  11. Glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050 )
  12. Neurobasal-A (Invitrogen, catalog number: 10888-022 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10888-022”.
  13. 1x Phosphate-buffer saline (PBS) (Roche Diagnostics, catalog number: 11-666-789-001 )
  14. 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15710-S )
  15. Monoclonal Anti-β-Tubulin III antibody produced in mouse (msIgG2b) (Sigma-Aldrich, catalog number: T8660 )
  16. Goat anti-mouse IgG2b 594 (Invitrogen, catalog number: A21145 )
    Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: A21145”.

Equipment

  1. 37 °C, 5% CO2 forced air incubator
  2. Dissecting microscope
  3. Surgical tools
    1. Forceps (Fine Science Tools, catalog number: 11254-20 )
    2. Microscissors (Fine Science Tools, catalog number: 15003-08 )
    3. Rongeurs (Fine Science Tools, catalog number: 16021-14 )
  4. Pipettes
  5. Hemocytometer
  6. Microcentrifuge

Procedure

  1. Prepping coverslips
    1. Place 12 mm glass coverslips in a 24-well plate (1 coverslip per well).
    2. Coat the coverslips with poly-l-lysine (0.1 mg/ml; PLL) either overnight at room temperature or for 2 h at 37 °C.
    3. Remove PLL from the coverslips in the wells.
    4. Add ddH2O to each well to wash the coverslips.
    5. Remove ddH2O using a pipette or suction. Do not allow the coverslips to dry completely. Upon removing ddH2O from the coverslips, immediately move to the next step.
    6. Bath coverslips in laminin (5 μg/ml) in HBSS-CMF and incubate at 37 °C for 2 h before plating cells.
      Notes:
      1. This concentration of laminin can be varied to enhance or reduce outgrowth potential.
        While dissociated adult DRG neurons grow well at this concentration, longer outgrowth can be obtained with higher laminin concentrations, whereas growing neurons on lower laminin concentrations will provide a more challenging substrate for outgrowth.
      2. Laminin should remain on coverslips until cells are ready to plate. There is no need to wash the coverslips once the laminin is removed.
      3. Neurons may be plated directly on laminin-coated coverslips, or plated on a monolayer of cells to examine the effects of neurite outgrowth on a cellular substrate.
    7. Plating a monolayer of cells
      1. A monolayer of cells can be created by densely plating a cell type of interest (astrocytes, stem cells, etc.) onto the coverslip after the laminin-coating has occurred.
      2. To create a monolayer, concentrate 60,000 cells/60 μl of media. (This concentration can be greater or less depending on the rate of cell division for a particular cellular population.)
      3. Remove laminin from the coverslips (step A6).
      4. Place the entire 60 μl drop of cells into the center of the coverslip and allow the plate to remain untouched at RT for 30 min. This will allow the cells some time to adhere to the coverslip.
      5. After the 30 min incubation, gently add an additional 1 ml of media down the side of the well so to minimize the dispersal or disruption of the cell solution in the center of the slip.
      6. Allow the cells to incubate overnight at 37 °C before adding DRG neurons.

  2. Dissociating DRG neurons
    1. Remove all DRGs from an adult mouse or rat.
    2. Trim the central and peripheral roots.
    3. Place DRGs into a 1.5 ml microcentrifuge tube containing collagenase II (200 U/ml) and dispase II (2.5 U/ml) in HBSS-CMF. Dispase/collagenase helps to digest the dura surrounding the ganglia, allowing the cell bodies to fully dissociate from one another.
    4. Incubate for 1 h at 37 °C.
    5. Remove DRGs from the incubator and centrifuge the sample for 2 min, 5,600 RPM.
    6. Gently remove supernatant and discard. Be careful not to disturb the pellet containing the cells.
    7. Add fresh CMF solution (~1 ml) to the microcentrifuge tube containing the pellet, gently triturate, then centrifuge for 2 min, 5,600 RPM.
    8. Repeat steps B6-7 two more times (3 total washes).
    9. After the third wash, resuspend the dissociated neurons in 1 ml of Neurobasal-A media supplemented with B-27, GlutaMAX, and penicillin-streptomycin.
      Note: Once the cells have been dissociated (step B3), it is important to complete the subsequent steps in a timely fashion (it should take approximately 20 min) to minimize neuronal death during the washes. If necessary, keep the cells on ice during this process.
    10. Using a hemocytometer, count the number of neuronal cell bodies present in the sample.
    11. Plate neurons on coverslips at a concentration of 1,500-2,000 neurons per coverslip (in 1 ml Neurobasal-A per coverslip).
      Note: Neurons can be plated on laminin-coated coverslips 2 h after coating them. Typically adding laminin just before beginning the DRG isolation and dissociation will allow plenty of time for the substrate to adhere to the coverslip.
    12. Incubate at 37 °C for 24 h.

  3. Plating cells for neurite outgrowth
    1. 24 h after DRG plating, remove the media from the wells.
    2. Add ~500 ml 4% PFA for 30 min at room temperature.
    3. Remove PFA.
    4. Wash 3 x 10 min with 1x PBS.
    5. Block coverslips overnight at 4 °C.
    6. Stain cultures with β-III tubulin (1:500 in block + triton) overnight at 4 °C or 2 h at room temperature.
    7. Wash cultures 3 times for 10 min each with 1x PBS.
    8. Stain with the appropriate secondary antibody overnight at 4 °C or 2 h at room temperature.
    9. Mount coverslips onto slides.
      1. To mount coverslips, use a pair of size 3 jeweler’s forceps to carefully lift the coverslip up the side of the well. Be sure to keep track of which side of the coverslip contains the cells.
      2. Carefully grab the edge of the coverslip with the forceps. Be careful not to grab the center of the coverslip or you will scrape off the cells.
      3. Flip the coverslip to be side down and set the coverslip onto the slide.
      4. Seal the edge of the coverslip with clear nail polish and allow the slides to dry.
    10. Image all neurons with processes.

  4. Measuring neurite outgrowth
    1. Using MetaMorph software, measure the longest neurite per neuron. This can be done using either the traced line or multi-line tool.
      1. Open MetaMorph software.


        Figure 1. Open screen

      2. To measure a neurite, open “Region Measurements.”


        Figure 2. Region measurements cropped

      3. The “Region Measurement” window should open. Select “active region” to see the length of the most recent neurite measured.


        Figure 3. Active region

      4. Go to “Measure.” Click “Calibrate Distances.”


        Figure 4. Calibrate distance

        Note: Distance may need to be calibrated based on the scope and magnification used to image the neurons. A calibration must be established for each objective used. These calibrations will vary based on scope. Therefore, for every scope and every objective, a calibration must be determined and added to the list.
        Select the calibration that corresponds to the appropriate microscope and objective used to image the neuron being measured.


        Figure 5. Calibrate distances screen

      5. Open the image to be measured.


        Figure 6. Image opened

      6. Using either the traced or multi-line tool, trace a process, beginning near the cell body, extending to the tip of the process. Processes often branch, so be sure to follow the appropriate path (the longest process).


        Figure 7. Traced tool


        Figure 8. Multi-line tool

      7. Once the entire process is traced, double click. This will display the “region measurement” or the length of the neurite that has just been traced. It will be displayed both on the image, as well as on the region measurements window.


        Figure 9. Begin clicking

        Be sure “Active Region Measurements” is displayed in the “Region Measurements” window to see the measurement of the most recently measured neurite.


        Figure 10. Active region


        Figure 11. Finish measurement

Notes

To determine the longest neurite per neuron, often multiple neurites per neuron will have to be measured, then select the longest of those measured. Depending on the density and placement of neurons, it may be difficult to determine whether a particular neurite is coming from one neuronal cell body or the other. If the cell body of origin cannot be determined, exclude those neurites/neurons from analysis.

Acknowledgements

This protocol was used in Filous et al. (2014). This work was supported by National Institutes of Health- National Institute of Neurological Disorders and Stroke Grant NS025713, National Institutes of Health Training Grant T32, and the New York State Department of Health and Brumagin Memorial Fund.

References

  1. Filous, A. R., Tran, A., Howell, C. J., Busch, S. A., Evans, T. A., Stallcup, W. B., Kang, S. H., Bergles, D. E., Lee, S. I., Levine, J. M. and Silver, J. (2014). Entrapment via synaptic-like connections between NG2 proteoglycan+ cells and dystrophic axons in the lesion plays a role in regeneration failure after spinal cord injury. J Neurosci 34(49): 16369-16384.

材料和试剂

  1. 12毫米玻璃盖片
  2. 24孔板
  3. 移液器提示
  4. 聚-L-赖氨酸氢溴酸盐(PLL)(Sigma-Aldrich,目录号:P1274)
  5. 层粘连蛋白小鼠蛋白质(Invitrogen,目录号:23017-015) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:23017-015"。
  6. HBSS-CMF(HBSS)(Thermo Fisher Scientific,Gibco TM ,目录号:14185-052)
  7. 胶原酶II(Worthington Biochemical Corporation,目录号:4176)
  8. Dispase II(Roche Diagnostics,目录号:04942078001)
  9. 青霉素 - 链霉素(Invitrogen,目录号:15140122) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140122"。
  10. B-27 无血清补充剂(50x)(Invitrogen,目录号:17504-044)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:17504-044"。
  11. Glutamax(Thermo Fisher Scientific,Gibco TM ,目录号:35050)
  12. Neurobasal-A(Invitrogen,目录号:10888-022) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:10888-022"。
  13. 1×磷酸盐缓冲盐水(PBS)(Roche Diagnostics,目录号:11-666-789-001)
  14. 4%多聚甲醛(PFA)(Electron Microscopy Sciences,目录号:15710-S)
  15. 小鼠产生的单克隆抗β微管蛋白III抗体(msIgG2b)(Sigma-Aldrich,目录号:T8660)
  16. 山羊抗小鼠IgG2b 594(Invitrogen,目录号:A21145) 注意:目前,"Thermo Fisher Scientific,Novex TM ,目录号:A21145"。

设备

  1. 37℃,5%CO 2强制空气培养箱
  2. 解剖显微镜
  3. 外科工具
    1. 镊子(Fine Science Tools,目录号:11254-20)
    2. Microscissors(Fine Science Tools,目录号:15003-08)
    3. Rongeurs(Fine Science Tools,目录号:16021-14)
  4. 移液器
  5. 血细胞计数器
  6. 微量离心机

程序

  1. 预先盖片
    1. 将12毫米玻璃盖玻片放在24孔板(每孔1盖玻片)
    2. 用聚-l-赖氨酸(0.1mg/ml; PLL)将盖玻片涂覆在室温下过夜或在37℃下2小时。
    3. 从孔中的盖玻片上取下PLL。
    4. 向每个孔中加入ddH 2 O以洗涤盖玻片
    5. 使用移液管或吸力管移除ddH 2 O。不要让盖玻片 ?完全干燥。一旦从盖玻片除去ddH 2 O,立即 ?移至下一步。
    6. 将层粘连蛋白(5μg/ml)在HBSS-CMF中的盖玻片孵育并在37℃孵育2小时,然后铺板细胞。 注意:
      1. 可以改变层粘连蛋白的浓度以增强或减少生长潜力。
        虽然解离的成年DRG神经元在该浓度下生长良好, 可以用更高的层粘连蛋白浓度获得更长的生长, 而增长的神经元对较低的层粘连蛋白浓度将提供 更具挑战性的底物用于生长。
      2. 层粘连蛋白应保留 在盖玻片上,直到细胞准备好平板。没有必要洗 一旦层粘连蛋白被去除后,盖玻片。
      3. 神经元可以镀 ?直接在层粘连蛋白包被的盖玻片上,或电镀在单层上 细胞以检查神经突向外生长对细胞的影响 底物。
    7. 电镀单层细胞
      1. 单层的 可以通过对感兴趣的细胞类型进行致密铺板来产生细胞 (星形胶质细胞,干细胞,等)到盖玻片上 发生层粘连蛋白涂层。
      2. 要创建单层,浓缩 ?60,000个细胞/60μl培养基。 (该浓度可以更大或更小 ?这取决于特定细胞的细胞分裂速率 人口。)
      3. 从盖玻片上去除层粘连蛋白(步骤A6)。
      4. 把整个60微升的细胞滴到盖玻片的中心 并使板在RT下保持不变30分钟。这会 允许细胞一些时间粘附到盖玻片。
      5. 之后 30分钟孵育,轻轻地添加额外1毫升培养基下侧 以便最小化细胞的分散或破裂 溶液在滑移的中心。
      6. 允许细胞在37°C孵育过夜,然后加入DRG神经元。

  2. 解离DRG神经元
    1. 从成年小鼠或大鼠中取出所有DRG
    2. 修剪中央和外围根。
    3. 将DRGs放入含有胶原酶的1.5ml微量离心管中 ?II(200U/ml)和分散酶II(2.5U/ml)的HBSS-CMF。 Dispase /胶原酶有助于消化神经节周围的硬脑膜, 允许细胞体彼此完全解离。
    4. 在37℃下孵育1小时。
    5. 从培养箱中取出DRG,离心样品2分钟,5600 RPM
    6. 轻轻除去上清液并丢弃。小心不要打扰含有细胞的沉淀。
    7. 加入新鲜CMF溶液(约1毫升)到微量离心管 含有沉淀,轻轻研磨,然后离心2分钟, 5,600 RPM。
    8. 重复步骤B6-7两次(总共洗涤3次)。
    9. 第三次洗涤后,将解离的神经元重悬在1ml的 补充有B-27,GlutaMAX和G-的Neurobasal-A培养基 青霉素 - 链霉素 注意:细胞解离后 (步骤B3),重要的是及时完成后续步骤 时尚(它应该需要大约20分钟),以尽量减少神经元死亡 ?在洗涤期间。如果需要,在此期间保持细胞在冰上 过程。
    10. 使用血细胞计数器,计数样品中存在的神经元细胞体的数量
    11. 在盖玻片上以每个盖玻片1,500-2,000个神经元的浓度(在每个盖玻片1ml Neurobasal-A中)平板神经元。
      注意:神经元可以在2小时后涂覆在层粘连蛋白包被的盖玻片上 涂层他们。通常在开始DRG之前添加层粘连蛋白 分离和解离将允许用于衬底的大量时间 以坚持盖玻片。
    12. 在37℃孵育24小时。

  3. 电镀细胞用于神经突增生
    1. DRG电镀24小时后,从孔中取出培养基
    2. 在室温下加入?500ml 4%PFA 30分钟
    3. 删除PFA。
    4. 用1x PBS洗涤3×10分钟。
    5. 在4℃封闭盖玻片过夜。
    6. 用β-III微管蛋白(在块+ triton中1:500)在4℃下培养过夜或在室温下2小时。
    7. 用1x PBS洗涤培养物3次,每次10分钟。
    8. 用适当的二抗在4℃下染色过夜或在室温下染色2小时。
    9. 将盖玻片装载到载玻片上。
      1. 要安装盖玻片,使用一对尺寸3的珠宝商的镊子 小心地将盖玻片从井的侧面提起。一定要保留 轨道的盖玻片的哪一侧包含细胞
      2. 用镊子小心地抓住盖玻片的边缘。小心 不抓住盖玻片的中心或你会刮掉 细胞
      3. 翻转盖玻片面朝下,将盖玻片放置在幻灯片上。
      4. 用清晰的指甲油密封盖玻片的边缘,让载玻片干燥。
    10. 图像所有神经元与进程。

  4. 测量神经突生长
    1. 使用MetaMorph软件,测量每个神经元的最长神经突。这个 可以使用跟踪线或多线工具。
      1. 打开MetaMorph软件。


        图1.打开屏幕

      2. 要测量神经突,打开"区域测量"

        图2.裁剪区域测量

      3. "区域测量"窗口应打开。选择"活动区域"以查看最近测量的神经突的长度。


        图3.活动区域

      4. 转到"测量"。单击"校准距离"。


        图4.校准距离

        注意:距离可能需要根据范围和 放大用于图像神经元。必须进行校准 建立了每个使用的目标。这些校准将基于不同 范围。因此,对于每个范围和每个目标,校准 必须确定并添加到列表中。
        选择与用于对正在测量的神经元成像的适当显微镜和物镜对应的校准

        图5.校准距离屏幕

      5. 打开要测量的图像。


        图6.图片已打开

      6. 使用跟踪或多线工具,跟踪过程, 在细胞体附近开始,延伸到过程的末端。 进程经常分支,所以一定要遵循适当的路径( 最长的过程)。


        图7.跟踪工具


        图8.多行工具

      7. 一旦 跟踪整个过程,双击。这将显示"区域 测量"或刚刚跟踪的神经突的长度。它 将显示在图像上以及区域上 测量窗口。


        图9.开始点击

        确保"活动区域 测量"显示在"区域测量"窗口中以查看 测量最近测量的神经突

        图10.活动区域


        图11.完成测量

笔记

为了确定每个神经元的最长神经突,通常每个神经元的多个神经元将必须被测量,然后选择那些测量的最长。根据神经元的密度和位置,可能难以确定特定神经突是来自一个神经元细胞体还是来自另一个神经元细胞体。如果不能确定来源的细胞体,从分析中排除那些神经突/神经元。

致谢

该协议在Filous等人使用。(2014)。这项工作得到国家卫生研究院 - 国家神经疾病和卒中拨款研究所NS025713,国立卫生研究院训练基金T32和纽约州卫生部和Brumagin纪念基金的支持。

参考文献

  1. Filous,A.R.,Tran,A.,Howell,C.J.,Busch,S.A.,Evans,T.A.,Stallcup,W.B.,Kang,S.H.,Bergles,D.E.,Lee,S.I.,Levine,J.M.and Silver, 通过NG2蛋白多糖 + 细胞和营养不良性轴突之间的突触样连接在损伤中在脊髓损伤后的再生衰竭中起作用。 J Neurosci 34(49):16369-16384。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Filous, A. R. and Silver, J. (2016). Neurite Outgrowth Assay. Bio-protocol 6(1): e1694. DOI: 10.21769/BioProtoc.1694; Full Text
  2. Filous, A. R., Tran, A., Howell, C. J., Busch, S. A., Evans, T. A., Stallcup, W. B., Kang, S. H., Bergles, D. E., Lee, S. I., Levine, J. M. and Silver, J. (2014). Entrapment via synaptic-like connections between NG2 proteoglycan+ cells and dystrophic axons in the lesion plays a role in regeneration failure after spinal cord injury. J Neurosci 34(49): 16369-16384.





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    我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

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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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