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Neuron Culture from Mouse Superior Cervical Ganglion
小鼠颈上神经节中神经元的培养   

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Abstract

The rodent superior cervical ganglion (SCG) is a useful and readily accessible source of neurons for studying the mechanisms of sympathetic nervous system (SNS) development and growth in vitro. The sympathetic nervous system (SNS) of early postnatal animals undergoes a great deal of remodeling and development; thus, neurons taken from mice at this age are primed to re-grow and establish synaptic connections after in situ removal. The stereotypic location and size of the SCG make it ideal for rapid isolation and dissociation. The protocol described here details the requirements for the dissection, culture and differentiation of SCG neurons. The protocol is suitable for culturing neurons from late embryonic gestation to approximately postnatal day 3. The culture technique discussed below utilizes glass coverslips for the microscopic examination of fixed cells.

Materials and Reagents

  1. Female mouse at desired gestational stage or early postnatal pups
  2. Sterile PBS (Corning Cellgro®, catalog number: 21-040-CV )
  3. L15 Leibovitz media (Corning Cellgro®, catalog number: 10-045-CV )
  4. Collagenase, Type 4 (1 mg) (Worthington Biochemical, catalog number: LS004182 )
  5. Collagenase enzyme solution (10 mg/ml in L15, filter-sterilized)
  6. Trypsin, 0.25% EDTA, Mg2+ Ca2+-free (Corning Cellgro®, catalog number: 25-053-Cl )
  7. Dulbecco’s Modified Eagle Medium (DMEM) (Corning Cellgro®, catalog number: 10-013-CV )
  8. Fetal bovine serum (FBS) (Equitech-Bio)
  9. Poly-D-lysine(PDL) (50 mg) (Sigma-Aldrich, catalog number: P0296 )
  10. Boric acid (Sigma-Aldrich, catalog number: B7660 )
  11. Sodium Tetraborate (Sigma-Aldrich, catalog number: B9876 )
  12. Laminin (1 mg) (BD Biosciences, catalog number: 354232 )
  13. 2.5 s Nerve Growth Factor (100 μg) (BD Biosciences, catalog number: 356004 )
  14. Concentrated nitric acid (Fisher Scientific, catalog number: A200-212 )
  15. Penicillin/streptomycin mix (Life Technologies, catalog number: 15140-122 )
  16. Sterile, deionized water
  17. Cytosine arabinoside (AraC) (Sigma-Aldrich, catalog number: C6645 )
  18. 0.1 M borate buffer (pH 8.5) (see Recipes)
  19. Regular plating medium (see Recipes)

Equipment

  1. Tissue culture incubator
  2. 35 mm or 6-well TC plates
  3. 35 mm tissue culture treated Petri dishes
  4. 100 mm Petri dishes
  5. 150 mm plastic Petri dish
  6. German glass coverslips, 25 mm (Electron Microscopy Sciences, catalog number: 72196-25 )
  7. Ceramic racks (Thomas Scientific, catalog number: 8542E40 )
  8. Basic gravity convection oven (VWR International, catalog number: 414005-108 )
  9. Silicon rubber dissection plates
  10. Scissors
  11. Fine tipped forceps
  12. 26 gauge needles
  13. Fire-polished, cotton-plugged, siliconized Pasteur pipets or Barrier tip 200
  14. Reduced bore siliconized Pasteur pipets
  15. Serological pipet
  16. Stereoscopic microscope
  17. Fume hood
  18. Water bath
  19. 37 °C incubator

Procedure

Note: All solutions and equipment coming into contact with cells must be sterile. Aseptic technique is critical throughout the procedure; thus, dissection taking place in a tissue culture hood modified for the use of a stereoscopic microscope may yield the best results.

  1. Preparation of glass coverslips
    1. Place coverslips in ceramic racks and rinse with water. Clean in concentrated nitric acid for 48 h in fume hood.
    2. Remove coverslips from acid and soak in MilliQ water for 1 h. Repeat three times.
    3. Cover racks in foil and place in hot air sterilization oven at 200 °C overnight.
    4. Place coverslips together in one 150 mm plastic Petri dish. Add ~50 ml of a filter-sterilized solution of 1 mg/ml poly-D-lysine (PDL) in borate buffer and leave overnight at RT.
    5. Rinse with sterile MilliQ water for 1 h. Repeat three additional times.
    6. Transfer individual coverslips to 35 mm or 6-well TC plates. Let dry completely.
      Note: Dried PDL-coated slips can be stored for weeks at 4 °C in sterile conditions.
    7. Apply ~400 μl laminin (10 μg/ml in sterile PBS) to coverslips intended for immediate use and incubate at 4 degrees until plating (≥ 4 h). Ensure that laminin doesn’t wick off the slips by centering them in the plates. 30 min before plating cells, place coverslips with laminin in tissue culture hood to bring to room temperature. Rinse slips 3 times with sterile PBS at room temperature. Do not allow coating to dry before plating neurons.

  2. Dissection
    1. Euthanize pregnant female and remove string of embryos or gather postnatal pups and euthanize in accordance with the requirements of your Institutional Animal Care and Use Committee. Place the pups or embryos in 100% ethanol to sterilize and then transfer to sterile PBS. In the case of embryos, placenta and other tissue can be removed in PBS. Allow pups or embryos to dry in a sterile 100 mm Petri dish.
    2. Decapitate pups/embryos into L15 remembering to clip the tails and reserve for later digestion if genotyping of animals is desired. The SCG are located in the bifurcation of the carotid arteries bilaterally coursing through neck region of the animal; decapitate animals as close to the “shoulder” area as possible to ensure full capture of the SCG (Figure 1A).
    3. Place the head with the cut surface up on a sterilized rubber dissection plate. Pin the head in place using two 26 gauge needles and flush with sterile PBS to clear the dissection field for viewing (Figure 1B).
    4. Under a stereoscopic microscope, locate the remnant of the common carotid artery and follow it deep into the head until the bifurcation is clearly seen. The SCG is stereotypically found at this junction between the internal and external carotid arteries and can be distinguished as a white to translucent, discrete spindle-shaped mass (Figure 1C-D). Often a large nerve can be seen branching from the superior aspect.
    5. Scoop beneath the SCG with fine forceps to gently remove the ganglion in one piece and place in a labeled plate with L15.
    6. Remove any excess tissue and debris from ganglia and place at 4 °C until dissociation.
      Note: Intact SCG can be stored in sterile conditions for up to 4 days at 4 °C in L15 without appreciable loss in subsequent quality of culture.

                               
      Figure 1. Dissection of the superior cervical ganglion from a postnatal day 0 (P0) mouse pup. A. Whole P0 pup anesthetized and sterilized before dissection. Dotted line shows approximate level for decapitation. B. Inferior aspect of the isolated head. The cut trachea (arrowhead) is visible just superior to the bilateral zones (asterisk) where the carotid arteries will be located once surrounding tissue is removed. C. The trachea has been displaced upward and excess tissue removed from the dissection field, exposing the SCG (dotted outline). D. Image showing two dissected ganglia with small remnants of surrounding tissue attached.  Scale bars = 1 mm (B, C, and D).

  3. Dissociation and plating
    1. Place SCG into ~500 μl collagenase enzyme solution for 30 min in 37 °C water bath. Agitate every 5-10 min.
    2. Remove collagenase and replace with 500 μl pre-warmed 0.25% trypsin. Place in 37 °C water bath for 15-20 min, agitating every 5 min.
    3. Remove trypsin and rinse ganglia 3 times in L15.
    4. Replace L15 with approximately 500 μl pre-warmed regular plating medium. Triturate with reduced bore siliconized Pasteur pipets. 60-70 times should be sufficient if single cells are desired.
    5. Transfer 500 μl of the dissociated neuron suspension to uncoated 35 mm Petri dish, keeping drop in center of plate. This pre-plating step assists in the removal of glial cells before culturing the neurons. Glial cells will adhere to the uncoated dish while neurons will remain in suspension.  Incubate 1 h in 37 °C incubator.
    6. Recover ~500 μl of the cell suspension with pipet tip. Dish can be gently tapped on the side to ensure good recovery of dissociated cells. Neurons can be spun down at 1,000 rpm for 10 min and re-suspended in pre-warmed medium as needed for high density (e.g., 2 SCG/70 μl for a 35 mm plate) or low density (e.g., 2/3-1 SCG/70 μl for a 35 mm plate) plating.
    7. After re-suspension, plate the cells on poly-D-lysine/laminin coated coverslips in 35 mm dishes or 6-well plates. Allow the neurons to adhere to the glass for 1.5-2 h in the incubator, then add 1.5-2 ml pre-warmed medium gently down the side of the dish with a serological pipet (dropwise). Incubate in 37 °C/5% CO2.
    8. If long-term culture is desired, new medium supplemented with 2 μM cytosine arabinoside (AraC) can be added at 3 days in vitro (DIV). Change the medium every 2-4 days thereafter to eliminate non-neuronal cells. Neurons can be cultured up to 28 DIV.


      Figure 2. Image of cultured neurons of the superior cervical ganglion. Representative image showing neuronal class III β-tubulin (Tuj1) labeled SCG neurons at 48 h post-plating.

Recipes

  1. 0.1 M Borate buffer
    Reagents                     Volume
    Boric acid                     1.24 g
    Sodium tetraborate      1.90 g
    dH2O                            400 ml
    pH 8.5, filter-sterilize
  2. Regular plating medium
    Reagents                                Volume
    FBS                                         5 ml
    Penicillin/streptomycin  mix     500 μl
    DMEM                                     45 ml
    Note: Nerve growth factor (NGF) is required for the growth and survival of sympathetic neurons in culture and should be added to plating medium. 100 ng/ml is suitable for signaling/long experiments; 10 ng/ml supports the survival of approximately 90% of neurons.

Acknowledgments

This protocol is adapted from Quach et al. (2013) and Eldredge et al. (2008).

References

  1. Eldredge, L. C., Gao, X. M., Quach, D. H., Li, L., Han, X., Lomasney, J. and Tourtellotte, W. G. (2008). Abnormal sympathetic nervous system development and physiological dysautonomia in Egr3-deficient mice. Development 135(17): 2949-2957.
  2. Quach, D. H., Oliveira-Fernandes, M., Gruner, K. A. and Tourtellotte, W. G. (2013). A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation. J Neurosci 33(10): 4570-4583.
  3. Xia, S., Lampe, P. A., Deshmukh, M., Yang, A., Brown, B. S., Rothman, S. M., Johnson, E. M., Jr. and Yu, S. P. (2002). Multiple channel interactions explain the protection of sympathetic neurons from apoptosis induced by nerve growth factor deprivation. J Neurosci 22(1): 114-122.

简介

啮齿动物上级子宫颈神经节(SCG)是一种有用且易于获取的神经元来源,用于研究交感神经系统(SNS)体外发育和生长的机制。 早期出生后动物的交感神经系统(SNS)经历了大量的重塑和发育; 因此,在这个年龄的小鼠中取出的神经元被引发再生长并在原位移除后建立突触连接。 SCG的刻板位置和尺寸使其成为快速隔离和解离的理想选择。 这里描述的方案详述了SCG神经元的解剖,培养和分化的要求。 该方案适用于从晚期胚胎妊娠至大约出生后第3天培养神经元。下面讨论的培养技术利用玻璃盖玻片进行固定细胞的显微镜检查。

材料和试剂

  1. 在所需的妊娠期或早期产后幼崽的雌性小鼠
  2. 无菌PBS(Corning Cellgro ,目录号:21-040-CV)
  3. L15 Leibovitz培养基(Corning Cellgro ,目录号:10-045-CV)
  4. 胶原酶,4型(1mg)(Worthington Biochemical,目录号:LS004182)
  5. 胶原酶酶溶液(10mg/ml,在L15中,过滤灭菌)
  6. 胰蛋白酶,0.25%EDTA,Mg 2+和Ca 2+的缓冲液(Corning Cellgro,目录号:25-053-Cl) br />
  7. Dulbecco改良的Eagle培养基(DMEM)(Corning Cellgro ,目录号:10-013-CV)
  8. 胎牛血清(FBS)(Equitech-Bio)
  9. 聚-D-赖氨酸(PDL)(50mg)(Sigma-Aldrich,目录号:P0296)
  10. 硼酸(Sigma-Aldrich,目录号:B7660)
  11. 四硼酸钠(Sigma-Aldrich,目录号:B9876)
  12. 层粘连蛋白(1mg)(BD Biosciences,目录号:354232)
  13. 2.5微克神经生长因子(100微克)(BD Biosciences,目录号:356004)
  14. 浓硝酸(Fisher Scientific,目录号:A200-212)
  15. 青霉素/链霉素混合物(Life Technologies,目录号:15140-122)
  16. 无菌,去离子水
  17. 阿糖胞苷(AraC)(Sigma-Aldrich,目录号:C6645)
  18. 0.1M硼酸盐缓冲液(pH 8.5)(参见配方)
  19. 常规电镀介质(参见配方)

设备

  1. 组织培养孵化器
  2. 35 mm或6孔TC板
  3. 35mm组织培养处理的培养皿
  4. 100 mm培养皿
  5. 150mm塑料培养皿
  6. 德国玻璃盖玻片,25mm(Electron Microscopy Sciences,目录号:72196-25)
  7. 陶瓷架(Thomas Scientific,目录号:8542E40)
  8. 基本重力对流烤箱(VWR International,目录号:414005-108)
  9. 硅橡胶夹层板
  10. 剪刀
  11. 细尖钳
  12. 26号针
  13. 火抛光,棉塞,硅化巴斯德吸管或阻挡尖端200
  14. 减少硅化的巴斯德移液管
  15. 血清移液器
  16. 立体显微镜
  17. 通风橱
  18. 水浴
  19. 37℃孵育器

程序

注意:所有与细胞接触的溶液和设备必须是无菌的。 无菌技术在整个过程中至关重要; 因此,在为使用立体显微镜而修改的组织培养罩中进行的解剖可以产生最好的结果

  1. 玻璃盖玻片的制备
    1. 将盖玻片放在陶瓷架中,用水冲洗。 在通风橱中在浓硝酸中清洁48小时。
    2. 从酸中取出盖玻片,在MilliQ水中浸泡1小时。 重复三次。
    3. 在箔中盖上架,并在200℃的热空气灭菌烘箱中放置过夜。
    4. 将盖玻片放在一起150毫米塑料培养皿。 加入〜50ml的在硼酸盐缓冲液中的1mg/ml聚-D-赖氨酸(PDL)的过滤灭菌的溶液,并在室温下放置过夜。
    5. 用无菌MilliQ水冲洗1小时。 重复三次。
    6. 将单个盖玻片转移到35毫米或6孔TC板。 让干完全。
      注意:干燥的PDL涂布的卡片可以在无菌条件下在4℃下储存数周。
    7. 应用〜400μl层粘连蛋白(10μg/ml在无菌PBS中)到盖玻片立即使用,并孵育在4度,直到电镀(≥4小时)。确保层粘连蛋白不会通过将它们居中在板中而芯吸。在铺板细胞前30分钟,将组织培养罩中的层粘连蛋白盖玻片放置至室温。在室温下用无菌PBS漂洗3次。在涂敷神经元之前不要让涂层干燥。

  2. 解剖
    1. 安乐死怀孕女性,并删除胚胎串或收集产后幼崽和安乐死,根据您的机构动物护理和使用委员会的要求。将幼崽或胚胎在100%乙醇中灭菌,然后转移到无菌PBS。在胚胎的情况下,可以在PBS中除去胎盘和其他组织。允许幼崽或胚胎在无菌的100mm培养皿中干燥
    2. 将幼崽/胚胎蜕变为L15,记住剪断尾巴,如果需要对动物进行基因分型,则留待以后消化。 SCG位于颈动脉的分叉处 双侧通过动物的颈部区域;将动物尽可能接近"肩"区域,以确保SCG的完全捕获(图1A)。
    3. 将头部与切割面放在无菌橡胶夹层板上。使用两个26号针将头部固定在适当位置,并用无菌PBS冲洗以清除解剖视野(图1B)。
    4. 在立体显微镜下,找到颈总动脉的残余,并深入头部,直到分叉清晰可见。 SCG在颈内动脉和外颈动脉之间的接合处是刻板式的,并且可以被区分为白色至半透明的,离散的纺锤形团块(图1C-D)。通常可以看到从上面分支的大神经。
    5. 在SCG下方用细镊子轻轻取出神经节,放置在带有L15的标记板上。
    6. 从神经节中取出任何多余的组织和碎片,并置于4°C直到解离 注意:完整的SCG可以在无菌条件下在4℃下在L15中保存长达4天,而后者的培养质量没有明显的损失。

                                  
      图1.来自出生后第0天(P0)小鼠幼仔的上颈部神经节的解剖。 A.整个P0小鼠在解剖前麻醉和灭菌。虚线表示断头的大致水平。 B.孤立头部的下方。切除的气管(箭头)可见于刚好在除去周围组织时颈动脉将位于的双边区(星号)的上方。 C.气管已经向上移位,并且从切开区域移除多余的组织,暴露SCG(虚线轮廓)。 D.图像显示两个解剖的神经节与周围组织的小残留物附加。比例尺= 1mm(B,C和D)
  3. 解离和电镀
    1. 将SCG置于〜500μl胶原酶酶溶液中37℃水浴中30分钟。每5-10分钟搅拌一次。
    2. 去除胶原酶,并更换500μl预热的0.25%胰蛋白酶。置于37℃水浴15-20分钟,每5分钟搅拌。
    3. 删除胰蛋白酶和漂洗神经节3次在L15。
    4. 用约500μl预热的常规电镀培养基替换L15。用还原孔硅化巴斯德吸管研磨。如果需要单细胞,60-70倍就足够了。
    5. 转移500微升解离的神经元悬浮液到未涂层35毫米培养皿,保持滴在板的中心。该预镀步骤有助于在培养神经元之前去除神经胶质细胞。胶质细胞将粘附于未涂布的皿,而神经元将保持悬浮。在37℃孵育1小时。
    6. 用移液管吸头回收〜500μl的细胞悬浮液。可以轻轻地轻拍侧面的菜,以确保解离的细胞的良好恢复。神经元可以以1,000rpm离心10分钟,并根据需要重新悬浮在预热的培养基中以获得高密度(例如,对于35mm平板为2SCG /70μl)或低密度,2/3-1SCG /对于35mm板的70μl)电镀。
    7. 重悬浮后,将细胞铺在聚-D-赖氨酸/层粘连蛋白包被的盖玻片上在35mm皿或6孔板中。允许神经元在培养箱中粘附玻璃1.5-2小时,然后用血清学移液管逐滴加入1.5-2 ml预热的培养基(用滴管)。在37℃/5%CO 2中孵育。
    8. 如果需要长期培养,可以在体外3天(DIV)添加补充有2μM阿糖胞苷(AraC)的新培养基。此后每2-4天更换培养基,以消除非神经元细胞。神经元可培养至28DIV

      图2. 上颈部神经节的培养神经元的图像。代表图像显示在铺板后48小时神经元III类β-微管蛋白(Tuj1)标记的SCG神经元。

食谱

  1. 0.1M硼酸盐缓冲液
    试剂                     卷
    硼酸                      1.24克
    四硼酸钠    1.90克
    dH 2 O                           400 ml
    pH 8.5,过滤灭菌
  2. 常规电镀介质
    试剂                            卷
    FBS                                       5 ml
    青霉素/链霉素mix    500微升
    DMEM                                    45 ml
    注意:神经生长因子(NGF)是培养中交感神经元的生长和存活所需的,并且应该加入到平板培养基中。 100ng/ml适合于信号传导/长实验; 10ng/ml支持大约90%的神经元的存活。

致谢

该协议改编自Quach等人(2013)和Eldredge等人(2008)。

参考文献

  1. Eldredge,L.C.,Gao,X.M.,Quach,D.H.,Li,L.,Han,X.,Lomasney,J.and Tourtellotte,W.G。(2008)。 Egr3缺陷小鼠的异常交感神经系统发育和生理性自主神经功能障碍。 Development 135(17):2949-2957。
  2. Quach,D.H.,Oliveira-Fernandes,M.,Gruner,K.A。和Tourtellotte,W.G。(2013)。 交感神经元自主角色的Egr3介导的基因调控在树突形态发生和目标组织神经支配。 a> J Neurosci 33(10):4570-4583。
  3. Xia,S.,Lampe,P.A.,Deshmukh,M.,Yang,A.,Brown,B.S.,Rothman,S.M.,Johnson,E.M.,Jr.and Yu,S.P。(2002)。 多渠道相互作用解释保护交感神经元免于由神经生长因子剥夺诱导的凋亡。 J Neurosci 22(1):114-122。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jackson, M. and Tourtellotte, W. (2014). Neuron Culture from Mouse Superior Cervical Ganglion. Bio-protocol 4(2): e1035. DOI: 10.21769/BioProtoc.1035.
  2. Quach, D. H., Oliveira-Fernandes, M., Gruner, K. A. and Tourtellotte, W. G. (2013). A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation. J Neurosci 33(10): 4570-4583.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。