搜索

Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn
小鼠脊髓背角神经元的原代培养   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular disease models, such as pain and itch. Because of the specific features of SDHN, it is necessary to establish a reliable culture method that is suitable for testing neural response to various external stimuli in vitro.

Keywords: Neuron(神经元), Culture(培养), Spinal cord(脊髓), Dorsal horn(背角), Pain(疼痛), Itch(瘙痒), Mouse (小鼠)

Background

Unlike existing protocols for culturing isolated mice primary neurons from hippocampus or cerebral cortex, few methods of culturing SDHN in vitro have been reported. This protocol was mainly based on previously described methods (Hu et al., 2003; Hugel and Schlichter, 2000). Here we made a few modifications including reagents, recipes, dissection and described step-by-step procedures of the dissection and culture of primary SDHN from newborn mice. In this protocol, neurons were gained using the enzymatic (papain) digestion method from fresh spinal dorsal horn tissues directly. The culture of SDHN in vitro can be used for further experiments, such as electrophysiological recordings, immunocytochemistry, and Ca2+ imaging, which better support cell behaviors in the spinal cord.

Materials and Reagents

  1. Coverslips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 174950 )
  2. 24-well cell culture plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
  3. Cell culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 153066 )
  4. Sterile centrifuge tubes (5 ml and 50 ml)
  5. Cotton ball
  6. Pipette tip  
  7. Cell strainer (40 μm) (Corning, Falcon®, catalog number: 352340 )
  8. Ice
  9. 3 days-old mice
  10. Diethyl ether (Sigma-Aldrich, catalog number: 346136 )
  11. 75% ethanol
  12. Cytosine arabinoside (Sigma-Aldrich, catalog number: C6645 )
  13. 4% formaldehyde
  14. 5% goat serum
  15. MAP2 antibody (Sigma-Aldrich, catalog number: M1406 )
  16. Anti-mouse FITC-conjugated secondary antibody
  17. DAPI (Sigma-Aldrich, catalog number: M9542 )
    Note: This product has been discontinued.
  18. Collagen (Sigma-Aldrich, catalog number: C7661 )
  19. Poly-D-lysine (Sigma-Aldrich, catalog number: P7405 )
  20. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  21. HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14025092 )
  22. Papain (Worthington Biochemical, catalog number: LS003119 )
  23. Neuro basal (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
  24. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 1099141 )
  25. Heat-inactivated horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050070
  26. B27 (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  27. Glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  28. Penicillin/Streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  29. Collagen stock solution (see Recipes)
  30. Poly-D-lysine (PDL) stock solution (see Recipes)
  31. Coating solution (see Recipes)
  32. HBSS + HEPES solution (see Recipes)
  33. Papain solution (see Recipes)
  34. Culture media (see Recipes)

Equipment

  1. Surgical scissors (decapitation/dissecting spinal column from body) (RWD Life Science, catalog number: S14014-14 )
  2. Blunt-tipped forceps (dissecting/holding spinal column) (RWD Life Science, catalog number: F13017-12 )
  3. Long and narrow-tipped spring scissors (dissecting spinal cord/cutting nerve roots) (66 Vision, catalog number: 54053B )
  4. Small-tipped spring scissors (cutting dura) (Fine Science Tools, catalog number: 15000-02 )
  5. Microforceps (holding spinal cord/trimming off dura) (World Precision Instruments, catalog number: 14098 )
  6. Scalpel #15 sterilized surgical blade (cutting spinal cord to isolate dorsal horn) (Shanghai Jinhuan Medical, catalog number: YY0174-2005 )
  7. Tissue culture hood (Suzhou Antai Airtech, model: SW-CJ-1F (D))
  8. Stereomicroscope (OLYMPUS, modle: SZ61 )
  9. Benchtop centrifuge (4 °C, 50 ml tube) (Eppendorf, model: 5430R )
  10. Pipette (Eppendorf, model: ES-1000 )
  11. Haemocytometer (Shanghai anxin optical instrument manufacture, model: XB-K-25 )
  12. Stackable CO2 incubator (Eppendorf, model: Galaxy®170R )

Procedure

  1. Select three 3 days-old mice.
  2. Coat coverslips with PDL/collagen/water coating solution.
    1. Put coverslips in a 24-well cell culture plate, with 1 piece/well. Add 500 μl coating solution in each well. This process is done under the hood.
    2. Incubate for 1-3 h in an incubator (37 °C).
    3. Transfer to the hood with cover off for 30 min to 1 h.
    4. Discard the excess solution, then let the coverslips air dry in the hood.
  3. Preparation (this process is done outside the hood in cell culture room)
    1. Place 5 small culture dishes (35 mm) filled with 4 ml ice-cold HBSS + HEPES solution on ice for dorsal horn tissue isolation.
    2. Prepare about 20 ml ice-cold HBSS + HEPES solution on ice for cell washes later.
    3. Preheat papain solution in 5 ml tube in the incubator for tissue digestion later.
    4. Preheat 20 ml aliquot of culture media in the incubator.
  4. Spinal cord dorsal horn dissection (Video 1, this process is done outside the hood in cell culture room). Add 1 ml Diethyl ether onto a cotton ball and put it into a 50 ml tube. Put the animal into the tube. Take it out after the animal stops moving. Spray the animal with 75% ethanol, decapitate with surgical scissors under deep diethyl ether anesthesia.

    Video 1. Dissection of the spinal cord dorsal horn

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    1. Cut the skin on the back and isolate the spinal column.
      1. Clamp the spinal column with blunt forceps.
      2. Pull up the column, cut the ribs lateral to the midline of spinal column and the ventral tissues attached to the column. Start rostrally and progress caudally.
      3. Make a transverse cut through the spinal column at the most caudal end and free the entire spinal column.
    2. Place the dissected spinal column into a culture dish with ice-cold HBSS + HEPES solution under the stereomicroscope. Arrange the column so that the ventral side is up and the rostral end is towards the right (Figure 1).


      Figure 1. The isolated spinal column. A shows the view of dorsal side. There are more attached muscles than the ventral side. B shows the view of ventral side. Ribs are attached to the column.

    3. Make ventral ‘laminectomy’
      1. Use large spring scissors to cut off the vertebral bones. Start from the rostral end and move towards the caudal end. Alternately cut the left side and the right side of the vertebrae. During the cutting, insert one blade of the scissors into the spinal canal and angle the scissors down and laterally away from the cord. Then use the tip of the scissors to lift up the ventral disk of the spinal column. Discard that chunk of vertebrae and the attached tissues (Figure 2).
      2. Repeat the procedure until the ventral surface of the spinal cord is exposed.


        Figure 2. Make ventral ‘laminectomy’. A and B. Cut off the vertebral bones from rostral to caudal side.

    4. Along the dorsolateral side of the cord, cut the connected nerve roots with large spring scissors. Always angle the scissors down and away from the cord (Figure 3). Gently free the cord from the spinal column with the closed tip of the spring scissors. Discard spinal column and other tissues.


      Figure 3. Cut the attached nerve roots (A) and isolate the cord (B)

    5. Place the cord into a new culture dish with ice-cold HBSS + HEPES solution. Put the rostral side of the cord forward (judge the caudal and rostral ends according to the direction of the nerve roots extended from the spinal cord) and the dorsal side is up (a blood vessel can be seen in the middle of the surface of the dorsal side) (Figure 4A).


      Figure 4. Isolate and cut the spinal cord. A. A blood vessel (marked by red dotted line) can be seen in the middle of the surface of the dorsal side. The direction (yellow arrows) of the nerve roots extended from the spinal cord to the caudal end. B. Cut the spinal cord along the midline, so that the medial side faces up. C. Cut approximately ¼ of dorsal part of spinal cord from caudal to rostral.

    6. Cut through the dura along the midline of the cord with small spring scissors.
      1. Use the closed tips of microforceps to gently press on the dorsal surface of the cord. (Make sure the cord is on the bottom of the dish, otherwise it will be difficult to manipulate and slice.)
      2. Cut from caudal end. While cutting, insert one blade (only the tip) of scissors under the dura and gently lift the dura to avoid destroying the cord.
      3. To cut ‘forward’ (rostral end), slide scissors rostrally while sliding microforceps caudally.
      4. Trim the dura attached to the dorsal surface of the cord with microforceps.
    7. Cut the spinal cord along midline (caudal-rostro axis) with scalpel (Figure 4B). Use the two tips of the microforceps to gently fix both sides of the cord and cut from the caudal to the rostral.
    8. Dissect the dorsal horn
      1. Take one half of the spinal cord and set the lateral side of the cord down and the medial side up (Figure 4B).
      2. Cut approximately ¼ of dorsal part of spinal cord from caudal to rostral.
      3. For the right hemisected cord, hold microforceps with your left hand and make careful cuts with scalpel held with right hand from caudal to rostral (Figure 4C).
      4. For the left hemisected cord, turn it around and cut from rostral to caudal.
      5. Discard the remaining ¾ of ventral spinal cord.
      6. Place dissected dorsal horn strip into a new culture dish with ice-cold HBSS + HEPES solution.

Note: Following steps are done in the hood.

  1. After collecting all the dorsal horn strips, transfer them to the 5 ml tube filled with the papain solution (step 3c).
  2. Digest for 30 min in the CO2 incubator at 37 °C (swirl every 5 or 10 min).
  3. Wash with 1-2 ml ice-cold HBSS + HEPES solution by aspirating liquid (do not aspirate the tissue). Put the tube stand for 2 min and let the tissue go down. Discard the liquid.
  4. Repeat washing twice.
  5. Wash the remaining tissue with 1 ml culture media.
  6. Discard as much liquid as possible and add another 1 ml culture media.
  7. Triturate for 6-7 times with a 1 ml fire-polished pipette tip (place the tip of pipette against wall of tube and aspirate the visible pieces up and down gently, avoid making any bubble) and then stand for 2-3 min.
  8. Collect about 600 μl supernatant and filter the collected supernatant with 40 μm cell strainer.
  9. Repeat steps 9-12 to collect about 3 ml in total and put the filtered supernatant into a 50 ml tube.
  10. Centrifuge at 1,000 x g for 5 min at 4 °C and remove as much supernatant as possible.
  11. Add 1 ml culture media to resuspend cells.
  12. Count cells using a hemacytometer and adjust the cell density to 5 x 105/ml with culture medium.
  13. Plate 200 μl supernatant onto the coated coverslips.
  14. Incubate for 60 min at 37 °C, then check for adherent cells and discard suspension cells, add 1 ml culture media to each well (for coverslips in 24-well plate).
  15. Change culture media on day 1 (replace half of the culture media), and then every 2 days (replace all).
  16. Two days after seeding, add cytosine arabinoside (10 μM, once) to the culture medium and maintain for 24 h to reduce glial proliferation.
  17. Use the cells for further experiments (Figure 5).


    Figure 5. Immunocytochemistry for evaluating the purity of the primary cultures of spinal dorsal horn neuron after 7 days. Double staining of MAP2 (neuronal marker, green) and DAPI (nuclear marker, blue) shows that majority of cultured cells are neurons.

Data analysis

To evaluate the purity of the primary cultures of spinal dorsal horn neuron, the double staining of immunofluorescence is used. Seven-days culture cells are fixed with 4% formaldehyde for 20 min and blocked with 5% goat serum for 2 h at room temperature. Then incubate overnight at 4 °C with mouse anti-MAP2 antibody (1:500) and incubate for 1 h at room temperature with anti-mouse FITC-conjugated secondary antibody (1:1,000; Jackson ImmunoResearch). After immunostaining, add 4,6-diamidino-2-phenylindole (DAPI) (0.1 g/ml) in the culture plates for 5 min at room temperature to stain all the nuclei of cells (Jiang et al., 2016).

Recipes

  1. Collagen stock solution
    Make up to 2 mg/ml in sterile double distilled water
    Aliquot and store at 4 °C
  2. Poly-D-lysine (PDL) stock solution
    Make up to 0.1 mg/ml in sterile double distilled water
    Aliquot and store at -20 °C
  3. Coating solution
    0.2 mg/ml collagen
    10 μg/ml Poly-D-lysine
    Dilute stock solutions (Recipes 1 and 2) with sterile double distilled water
  4. HBSS + HEPES solution
    Add 5 ml HEPES (1 M) to 500 ml HBSS and aliquot into 50 ml tubes
  5. Papain solution
    Add 45 μl papain to 3 ml HBSS + HEPES solution
  6. Culture media
    Neuro basal supplemented with 2% of fetal bovine serum, 2% of heat-inactivated horse serum, 2% of B27, 2 mM glutamax, and 1% of penicillin-streptomycin

Acknowledgments

Primary cultures of mouse spinal dorsal horn neuron protocol was funded by the National Natural Science Foundation of China (NSFC 31371121, 81400915, and 31671091). This protocol was developed and used in article published by Jiang et al., 2016.

References

  1. Hu, H. J., Glauner, K. S. and Gereau, R. W. t. (2003). ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. I. Modulation of A-type K+ currents. J Neurophysiol 90(3): 1671-1679.
  2. Hugel, S. and Schlichter, R. (2000). Presynaptic P2X receptors facilitate inhibitory GABAergic transmission between cultured rat spinal cord dorsal horn neurons. J Neurosci 20(6): 2121-2130.
  3. Jiang, B. C., Cao, D. L., Zhang, X., Zhang, Z. J., He, L. N., Li, C. H., Zhang, W. W., Wu, X. B., Berta, T., Ji, R. R. and Gao, Y. J. (2016). CXCL13 drives spinal astrocyte activation and neuropathic pain via CXCR5. J Clin Invest 126(2): 745-761.

简介

感觉神经元的主要传入主要终止于脊髓背角,其在感觉相关信号的整合和调节中具有重要作用。小鼠脊髓背角神经元(SDHN)的原代培养可用于研究从周围神经系统到脑的信号传递,以及用于发展诸如疼痛和瘙痒的细胞疾病模型。由于SDHN的具体特征,有必要建立一种可靠的培养方法,适合于测试体外各种外部刺激的神经反应。

背景 不同于目前用于培养分离的小鼠来自海马或大脑皮质的原代神经元的方案,报道了很少的在体外培养SDHN的方法。该协议主要基于以前描述的方法(Hu et al。,2003; Hugel和Schlichter,2000)。在这里,我们进行了一些修改,包括试剂,食谱,解剖和描述从新生小鼠的初步SDHN的解剖和培养的分步程序。在该方案中,使用来自新鲜脊髓背角组织的酶(木瓜蛋白酶)消化方法直接获得神经元。体外SDHN的培养可以用于进一步的实验,例如电生理记录,免疫细胞化学和Ca 2+成像,其更好地支持脊髓中的细胞行为。

关键字:神经元, 培养, 脊髓, 背角, 疼痛, 瘙痒, 小鼠

材料和试剂

  1. 盖板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:174950)
  2. 24孔细胞培养板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:142475)
  3. 细胞培养皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:153066)
  4. 无菌离心管(5ml和50ml)
  5. 棉球
  6. 移液器尖端
  7. 细胞过滤器(40μm)(Corning,Falcon ®,目录号:352340)

  8. 3日龄老鼠
  9. 乙醚(Sigma-Aldrich,目录号:346136)
  10. 75%乙醇
  11. 阿糖胞苷(Sigma-Aldrich,目录号:C6645)
  12. 4%甲醛
  13. 5%山羊血清
  14. MAP2抗体(Sigma-Aldrich,目录号:M1406)
  15. 抗小鼠FITC共轭二抗
  16. DAPI(Sigma-Aldrich,目录号:M9542)
    注意:本产品已停产。
  17. 胶原蛋白(Sigma-Aldrich,目录号:C7661)
  18. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P7405)
  19. HEPES(1 M)(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  20. HBSS(Thermo Fisher Scientific,Gibco TM ,目录号:14025092)
  21. 木瓜蛋白酶(Worthington Biochemical,目录号:LS003119)
  22. 神经基础(Thermo Fisher Scientific,Gibco TM ,目录号:10888022)
  23. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:1099141)
  24. 热灭活马血清(Thermo Fisher Scientific,Gibco TM,目录号:26050070) 
  25. B27(50x)(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  26. Glutamax(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  27. 青霉素/链霉素(100x)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  28. 胶原储备溶液(参见食谱)
  29. 聚-D-赖氨酸(PDL)储备溶液(参见食谱)
  30. 涂层溶液(见配方)
  31. HBSS + HEPES溶液(参见食谱)
  32. 木瓜蛋白酶溶液(参见食谱)
  33. 文化媒体(见食谱)

设备

  1. 手术剪刀(斩首/解剖脊柱从身体)(RWD生命科学,目录号:S14014-14)
  2. 钝镊子(解剖/保持脊柱)(RWD Life Science,目录号:F13017-12)
  3. 长而窄的弹簧剪刀(解剖脊髓/切割神经根)(66视力,目录号:54053B)
  4. 小型弹簧剪刀(切割硬脑膜)(精细科学工具,目录号:15000-02)
  5. Microforceps(控制脊髓/修剪硬膜)(世界精密仪器,目录号:14098)
  6. 手术刀#15灭菌手术刀(切脊髓隔离背角)(上海金环医疗,目录号:YY0174-2005)
  7. 组织文化罩(苏州安泰航空,型号:SW-CJ-1F(D))
  8. 立体显微镜(OLYMPUS,模式:SZ61)
  9. 台式离心机(4℃,50ml管)(Eppendorf,型号:5430R)
  10. 移液器(Eppendorf,型号:ES-1000)
  11. 血细胞计数器(上海安新光学仪器制造,型号:XB-K-25)
  12. 可堆叠的CO 2培养箱(Eppendorf,型号:Galaxy 170R)

程序

  1. 选择三只3只老鼠。
  2. 涂层盖玻片与PDL /胶原/水涂层溶液。
    1. 将盖玻片置于24孔细胞培养板中,每片1片。在每个孔中加入500μl涂层溶液。这个过程是在引擎盖下完成的
    2. 在孵育箱(37°C)孵育1-3小时。
    3. 转移到罩盖30分钟到1小时。
    4. 丢弃多余的溶液,然后让盖玻片在罩内空气干燥。
  3. 准备(这个过程在细胞培养室外面)
    1. 将5个装有4ml冰冷HBSS + HEPES溶液的小型培养皿(35mm)放在冰上进行背角组织分离。
    2. 在冰上准备约20毫升冰冷的HBSS + HEPES溶液进行细胞洗涤
    3. 预孵育后的木瓜蛋白酶溶液在5ml培养箱中进行组织消化
    4. 预热培养箱中20ml等分的培养基。
  4. 脊髓背角解剖(视频1,这个过程是在细胞培养室外面做的)。在棉球上加入1ml二乙醚,并将其放入50ml管中。把动物放进管子里。动物停止移动后取出。用75%乙醇喷洒动物,在深二乙醚麻醉下用手术剪刀斩首
    <! - flashid2098v158开始 - >
    视频1.脊髓背角解剖
    <! - [if!IE]> - > <! - <![endif] - >

    要播放视频,您需要安装较新版本的Adobe Flash Player。

    Get Adobe Flash Player

    - ! - [if!IE]> - >
    <! - <![endif] - >
    <! - flashid2098v158结束 - >
    1. 剪下背部皮肤,分离脊柱。
      1. 用钝钳钳住脊柱。
      2. 拉起柱子,将脊柱的中线和连接到柱的腹侧组织切开肋骨。从头开始,进步。
      3. 在最尾端穿过脊柱进行横切,释放整个脊柱。
    2. 将立体显微镜下将解剖后的脊柱放入含冰冷HBSS + HEPES溶液的培养皿中。排列柱,使腹侧朝上,朝向端向右(图1)。


      图1.孤立的脊柱。 A显示背侧视图。腹侧有更多的肌肉。 B显示腹侧视图。肋骨附在柱上。

    3. 腹侧"椎板切除术"
      1. 用大型剪刀切断椎骨。从头端开始,朝向尾端移动。替代切割椎骨的左侧和右侧。在切割过程中,将一把剪刀插入椎管内,使剪刀向下并横向离开绳索。然后使用剪刀的尖端抬起脊柱的腹盘。丢弃那块椎骨和附着的组织(图2)
      2. 重复该过程,直到脊髓的腹面露出

        图2.进行腹侧"椎板切除术"。 A和B.将脊骨从头端切断到尾侧。

    4. 沿着绳索的背侧,用大型剪刀切断连接的神经根。始终将剪刀向下并远离电线(图3)。用弹簧剪刀的封闭尖端将脊髓轻轻地从脊柱上释放。丢弃脊柱和其他组织。


      图3.切割附着的神经根(A)并分离绳索(B)

    5. 将电线放入带有冰冷HBSS + HEPES溶液的新培养皿中。将脊髓的前端放在前面(根据从脊髓伸出的神经根的方向判断尾部和尾端),背侧是上(在血管的表面中部可见血管背侧)(图4A)

      图4.隔离和切割脊髓 A.在背侧表面的中间可以看到血管(由红色虚线标记)。神经根的方向(黄色箭头)从脊髓延伸到尾端。 B.沿着中线剪下脊髓,使内侧面朝上。 C.将大约1/4的脊髓部分从尾部切割成流鼻血。

    6. 用小弹簧剪刀沿着绳索的中线穿过硬脑膜。
      1. 使用微型压缩机的封闭尖端轻轻按压电线背面。 (确保电线位于菜肴的底部,否则将难以操作和切片。)
      2. 从尾端切开在切割时,将剪刀下的一个刀片(只有尖端)插入硬脑膜,轻轻抬起硬脑膜,以免损坏电线。
      3. 切割"向前"(朝向末端),滑动剪刀,同时滑动微型锚杆。
      4. 用微型力量修剪附着在电线背面的硬膜。
    7. 用手术刀沿着中线(尾状罗斯托轴)切割脊髓(图4B)。使用微型推杆的两个尖端轻轻地固定绳索的两侧,并从尾部切割到长颈鹿。
    8. 解剖背角
      1. 取脊髓的一半,将腰部的侧面向下,内侧朝上(图4B)。
      2. 将大约1/4的脊髓背部切割成尾部。
      3. 对于正确的半切线,用左手握住微型手柄,并用右手从尾部到长柄手术,小心切开手术刀(图4C)。
      4. 对于左半球线,将其旋转并从头端切割到尾部。
      5. 丢弃腹侧脊髓的剩余¾
      6. 将解剖的背角条放入含有冰冷HBSS + HEPES溶液的新培养皿中

注意:以下步骤在引擎盖中完成。

  1. 收集所有背角条后,将其转移到装有木瓜蛋白酶溶液的5ml管中(步骤3c)。
  2. 在CO 2培养箱中37℃消化30分钟(每5或10分钟旋转)。
  3. 用1-2ml冰冷的HBSS + HEPES溶液通过吸液(不抽吸组织)洗涤。将管架放置2分钟,让组织下降。丢弃液体。
  4. 重复洗涤两次。
  5. 用1ml培养基洗涤剩余的组织。
  6. 尽可能多地丢弃液体,再添加1ml培养基
  7. 用1毫升火焰抛光的移液器尖端(将移液管的尖端放在管壁上,轻轻吸取可见的部分,避免产生任何气泡),然后静置2-3分钟,研磨6-7次。 br />
  8. 收集约600μl上清液,并用40μm细胞过滤器过滤收集的上清液。
  9. 重复步骤9-12,总共收集约3ml,将过滤的上清液放入50ml管中
  10. 在4℃下以1,000×g离心5分钟,并除去尽可能多的上清液。
  11. 加入1ml培养基以重悬细胞
  12. 使用血细胞计数器计数细胞,并用培养基将细胞密度调节至5×10 5/ml。
  13. 将200μl上清液放在涂覆的盖玻片上。
  14. 在37℃下孵育60分钟,然后检查贴壁细胞并丢弃悬浮细胞,向每个孔中加入1ml培养基(用于24孔板中的盖玻片)。
  15. 在第1天更换文化媒体(取代一半的文化媒体),然后每2天(全部更换)。
  16. 播种两天后,将阿糖胞苷(10μM,一次)加入到培养基中并保持24小时以减少胶质增生。
  17. 使用细胞进行进一步的实验(图5)

    图5.用于评估7天后脊髓背角神经元原代培养物的纯度的免疫细胞化学。MAP2(神经元标记,绿色)和DAPI(核标记,蓝色)的双重染色显示大多数培养的细胞是神经元

数据分析

为了评估脊髓背角神经元的原代培养物的纯度,使用免疫荧光的双染色。 7天培养细胞用4%甲醛固定20分钟,并在室温下用5%山羊血清封闭2小时。然后在4℃下用小鼠抗MAP2抗体(1:500)孵育过夜,并在室温下用抗小鼠FITC缀合的二抗(1:1,000; Jackson ImmunoResearch)孵育1小时。免疫染色后,在室温下,在培养板中加入4,6-二脒基-2-苯基吲哚(DAPI)(0.1μg/ml)5分钟,染色细胞的所有细胞核(Jiang等, 。,2016)。

食谱

  1. 胶原蛋白溶液
    在无菌双蒸水中补充2 mg/ml 等分并储存在4°C
  2. 聚-D-赖氨酸(PDL)储备液
    在无菌双蒸水中补充0.1 mg/ml 等分并储存于-20°C
  3. 涂层溶液
    0.2 mg/ml胶原蛋白
    10μg/ml聚-D-赖氨酸
    用无菌双蒸水稀释储备溶液(食谱1和2)
  4. HBSS + HEPES解决方案
    将5ml HEPES(1M)加入到500ml HBSS中,并分装到50ml管中
  5. 木瓜蛋白酶解决方案
    加入45μl木瓜蛋白酶至3 ml HBSS + HEPES溶液
  6. 文化媒体
    补充有2%胎牛血清,2%热灭活马血清,2%B27,2mM谷氨酸和1%青霉素 - 链霉素的神经基础

致谢

小鼠脊髓背角神经元方案的原代培养由中国国家自然科学基金资助(NSFC 31371121,81400915和31671091)。该协议是由Jiang等人发表的文章,2016年开发和使用的。

参考文献

  1. Hu,H.J.,Glauner,K.S。和Gereau,R.W。 (2003)。 ERK将PKA和PKC信号整合在表面背角神经元。 I.调制A型K + 电流。神经生物体90(3):1671-1679。
  2. Hugel,S.和Schlichter,R。(2000)。突触前P2X受体促进培养的大鼠脊髓背角神经元之间的抑制性GABA能传递。 Neurosci 20(6):2121-2130。
  3. 江,BC,曹,DL,张,X,张,ZJ,他,LN,李,CH,张,WW,吴,XB,Berta,T.,Ji,RR和高,YJ(2016) ; CXCL13通过CXCR5驱动脊髓星形细胞激活和神经性疼痛。 a> J Clin Invest 126(2):745-761。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cao, D., Jing, P., Jiang, B. and Gao, Y. (2017). Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn. Bio-protocol 7(1): e2098. DOI: 10.21769/BioProtoc.2098.
提问与回复

(提问前,请先登录)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。

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。