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Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons
小鼠背根神经节神经元的分离和原代细胞培养   

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Abstract

We here provide a detailed protocol for the isolation and culture of primary mouse sensory neurons. The cell bodies of sensory afferent pseudounipolar neurons are located in dorsal root ganglia (DRGs) along the vertebral column. Dissected mouse DRGs can be dissociated into single cells by enzymatic digestion to obtain primary cultures of mouse sensory neurons as performed in the studies reported by Khaminets et al. (2015).

Material and Reagents

  1. T25 cell culture flask (25 cm2) (Greiner Bio-One GmbH, catalog number: 690160 )
  2. 24-well plate (Greiner Bio-One GmbH, catalog number: 662160 )
  3. Cover glasses (Marienfeld-Superior, catalog number: 0111550 )
  4. Glass Pasteur pipettes (Marienfeld-Superior, catalog number: 3233050 )
  5. Filtropur S 0.2 syringe filters (Sarstedt AG, catalog number: 83.1826.001 )
  6. Syringe Omnifix® (B. Braun Medical, catalog number: 4616103V )
  7. Dissection dish (petri dish) (94 x 16 mm) (Sigma-Aldrich, catalog number: Z617636 ) with silicone pad
  8. Greiner Petri dishes (35 x 10 mm) (Sigma-Aldrich, catalog number: P5112 )
  9. 15 ml Cellstar centrifugation tubes (Greiner Bio-One GmbH, catalog number: 188261 )
  10. Mice (2 to 6 months) (strain: C57BL/6)
  11. Poly-L-lysine hydrobromide (Sigma-Aldrich, catalog number: P2636 )
  12. HEPES (AppliChem GmbH, catalog number: A3268 )
  13. H3BO3 (Carl Roth GmbH + Co., catalog number: 6943.1 )
  14. Sodium tetraborate (Na2B4O7) (Sigma-Aldrich, catalog number: 221732 )
  15. Bovine Serum Albumin (PAA, catalog number: K41-001 )
  16. HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175-053 )
  17. Neurobasal®-A medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
  18. B-27® Supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
  19. L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030 )
  20. Penicillin/streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
  21. Minimum Essential Medium (MEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31095-029 )
  22. D(+)-Glucose (C6H12O6) (Merck Millipore Corporation, catalog number: 108337 )
  23. Collagenase-II (Worthington Biochemical Corporation, catalog number: LS004176 )
  24. Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050-88 )
  25. Trypsin from bovine pancreas (Sigma-Aldrich, catalog number: T1426 )
  26. β-nerve growth factor (β-NGF) (Preprotec, catalog number: 450-01 )
  27. Borate buffer (see Recipes)
  28. Poly-L-lysine hydrobromide (PLL) stock solution (see Recipes)
  29. Poly-L-lysine hydrobromide (PLL) working solution (see Recipes)
  30. Dissociation solution (see Recipes)
  31. 30% Glucose stock solution (see Recipes)
  32. 0.5% BSA-blocking solution (see Recipes)
  33. DRG neuronal culture medium (see Recipes)
  34. DRG preparation medium (see Recipes)
  35. Collagenase-II solution (see Recipes)
  36. 2% trypsin stock solution (see Recipes)

Equipment

  1. CO2 incubator (37 °C, 5% CO2 concentration and 95% relative humidity) (Labotect, model: C-200 )
  2. Laminar flow work bench (Heraeus Holding)
  3. Stereo microscope (Carl Zeiss, model: Stemi 2000C )
  4. Cold light source (equipped with a flexible dual branch light guide) (Carl Zeiss, model: CL1500ECO )
  5. Scissors
    1. Standard scissors (Fine Science Tools, catalog number: 14002-12 )
    2. Student spring scissors (Fine Science Tools, catalog number: 91500-09 )
    3. Angled spring scissors (Fine Science Tools, catalog number: 15006-09 )
  6. Forceps
    1. Dumont #7b medical forceps (Fine Science Tools, catalog number: 11270-20 )
    2. Dumont #4 forceps (Fine Science Tools, catalog number: 11242-40 )
    3. Dumont #5 mirror finish forceps (Fine Science Tools, catalog number: 11252-23 )
  7. Bunsen burner Fireboy plus (Integra Biosciences)
  8. Glass Pasteur pipettes (Marienfeld-Superior, catalog number: 3233050 )
  9. Dropper bulb for Pasteur pipettes (Thermo Fisher Scientific, catalog number: 03-448-25 )
  10. Centrifuge 5810R (Eppendorf AG)
  11. Pipetman P20/P200/P1000 (Gilson)

Procedure

  1. Day 1: Dissociation solution is prepared and precooled to 4 °C.
  2. A T25 cell culture flask is coated with Poly-L-lysine hydrobromide (PLL). For this purpose 1 ml of PLL solution (1 mg/ml in borate buffer) is added to the cell culture flask and incubated overnight at 37 °C. Alternatively, eight cover glasses are placed into the wells of a sterile 24-well plate and incubated with PLL overnight at 37 °C.
  3. Day 2: PLL solution is aspirated and the cell culture flask is rinsed three times with sterile water. The flask is left open for drying in a laminar flow hood for 15 min.
  4. Then the flask is pre-incubated with 5 ml of DRG neuronal culture medium and placed into the incubator.
  5. Sterile glass pipettes are blocked by pipetting up and down 0.5% BSA-blocking solution three times. Blocked glass Pasteur pipettes are stored in the laminar flow work bench till usage at the same day.
  6. The scissors and forceps are disinfected for 30 min in 70% ethanol followed by air drying.
  7. The mouse is killed via cervical dislocation and the spinal cord is dissected rapidly with a pair of standard scissors. To prepare the spinal cord, the skin is incised at the ventral medial line with the standard scissors. Organs are removed from the thoracic and abdominal cavity. Using the standard scissors, two long cuts are made closely left and right to the spinal column. The tail and skull are removed. At last, remaining paravertebral muscles are largely removed to expose the spinal column.
  8. Using a stereo microscope, DRGs from all spinal levels are carefully removed and collected in 10 ml of HBSS in a 10-cm-dissecting dish placed on ice. In detail, the spine is cut first into three approximately equal pieces with the student spring scissors to improve subsequent handling. Then, one blade of the student spring scissors is inserted from the ventral side into the spinal canal. The other blade of the scissors is placed outside the spinal column on its ventral side. Two incisions are made along the spine, one left and one right of the midline on the ventral side as illustrated in Figure 1A-C and the scheme of Figure 2. The excised part of the spine is lifted and the spinal marrow is removed from the column (see Figure 1D-F). DRGs are clusters of somata of sensory neurons and are located closely along the dorsal root of the cord. DRGs are lying in the intervertebral foramina and have a long distal and proximal process (pseudounipolar neurons). DRGs are characterized by a round shape and hyaline appearance that differs from the white color of nerve fiber bundles. The student spring scissors, Dumont #7b and #4 forceps are used to carefully isolate the exposed ganglia for each of the three pieces of the spinal cord.
  9. Isolated DRGs are collected in a 35-mm-petri dish filled with 5 ml of DRG preparation medium. The dish is kept on ice. Approximately 20 to 40 ganglia are obtained according to the expertise of the experimenter.
  10. The thin layer of connective tissue of the epineurium surrounding the ganglion is removed with the help of a pair of spring scissors and the Dumont #5 Mirror Finish Forceps. Then the DRGs are transferred to a second 35-mm dish filled with DRG preparation medium on ice.
  11. Collagenase-II solution is prepared and preheated for 10 min to 37 °C.
  12. Isolated DRGs are transferred into a 15-ml-centrifugation tube with the help of the glass Pasteur pipette under a laminar flow workbench.
  13. After DRGs are settled on the tube bottom, the supernatant is removed and discarded.
  14. DRGs are rinsed with 5 ml of precooled and sterile dissociation solution.
  15. Again, the supernatant is removed. As described in steps 13 and 14, DRGs are washed three times in total, each with 5 ml of dissociation solution.
  16. After the last washing step, 2 ml of dissociation solution are left and 1 ml of Collagenase-II solution is added.
  17. DRGs are incubated for 1 h at 37 °C while gently shaking the Falcons every 10 min.
  18. An aliquot of the 2%-trypsin stock solution is defrosted and preheated for 20 min to 37 °C.
  19. 150 µl of activated trypsin is added.
  20. The DRGs are incubated for further 9 min at 37 °C while gently shaking the falcon every 3 min.
  21. The supernatant is removed carefully and washed with 5 ml of dissociation solution as described in steps 13 and 14.
  22. After the last washing step, 1.5 ml of dissociation solution are left.
  23. To obtain single cells, DRGs are dissociated with the glass Pasteur pipettes that had been prepared in step 5. Two glass Pasteur pipettes are fire-polished to obtain smaller openings. Using the glass pipette with an original opening diameter of 1.1-1.3 mm DRGs are pipetted approximately 10 times up and down till the solution appeared homogenous. Then the (fire-polished) Pasteur pipette with the smaller diameter is used and the solution triturated 8 times. Finally, the very small diameter (fire-polished) Pasteur pipette is used 3 times.
  24. The single cell suspension is centrifuged for 5 min at 160 x g. The deceleration of the centrifuge is reduced to ‘3’ (out of 10 braking ramps) in order to avoid that the small and fragile cell pellet gets dispersed while the centrifuge is stopped.
  25. The supernatant is removed from the cell pellet.
  26. DRG neuronal culture medium supplemented with 10% horse serum is added to the cell pellet; cells are resuspended and centrifuged for 5 min at 160 x g. The deceleration of the centrifuge is again set to ‘3’.
  27. The supernatant is carefully removed and cells are resuspended in DRG neuronal culture medium with a 1,000 µl pipette.
  28. Cells obtained from one mouse (approximately 50,000 to 100,000 cells) are plated in one T25 cell culture flask or alternatively on eight cover glasses (10 mm diameter) and placed at 37 °C under a 5% CO2 atmosphere.
  29. 30 min after plating, medium is replaced by fresh DRG cell culture medium supplemented with β-NGF (50 ng/ml). A volume of 5 ml is added to a T25 flask or 0.5 ml for each well of a 24-well plate.
  30. Primary cultures are maintained for up to three weeks. One third of the volume of DRG neuronal culture medium freshly supplemented with β-NGF (50 ng/ ml) is replaced every 3 to 5 days. Figure 3 shows bright field microscopy images of cultured DRG neurons at different time points after plating demonstrating the progressive growth of DRG neuronal processes in culture. Staining for the neuron-specific class III beta-tubulin (see Figure 4) further illustrates the characteristic morphology of cultured DRG neurons stimulated by β-NGF supplemented to the culture medium.

Representative data


Figure 1. Illustration of DRG dissection. A. The ventral side of the spinal cord is shown. B-C. Two incisions are made along the ventral side of the spinal cord, one left (B) and one right (C). D-E. The excised part of the spine is lifted. F. The spinal marrow is removed from the column. G. Ganglia are exposed and (H) carefully isolated from the surrounding tissue. I. The arrow points to an isolated dorsal root ganglion with its characteristic round shape, hyalin appearance and part of its proximal process.


Figure 2. Schematic illustration of spinal cord preparation for DRG isolation. For opening of the spinal canal two incisions are done (red dotted lines). The location of sensory ganglia at the dorsal side is shown.


Figure 3. Dissociated primary DRG neurons in culture. Brightfield images of primary mouse dorsal root ganglion neurons (A) three hours after plating, (B) at day 1 in vitro, (C) at day 2 in vitro and (D) at day 5 in vitro. Scale bars: 10 µm


Figure 4. Confocal images of fluorescently labeled primary DRG neurons. A. Primary mouse dorsal root ganglia neurons after 4 days in culture. Fixed cells are stained with neuron-specific class III beta-tubulin (Clone TUJ1, Stemcell technologies, 1:500). B. Nuclei are stained with Hoechst 33258. C. Merged image. Scale bars: 100 µm

Recipes

  1. Borate buffer
    1.24 g H3BO3
    1.90 g Na2B4O7
    Add ddH2O to 400 ml
    Stored at 4 °C for up to 1 year
  2. Poly-L-lysine hydrobromide (PLL) stock solution
    Dissolve 100 mg of PLL in 10 ml of ddH2O
    Aliquot and stored at -20 °C for up to 6 months
  3. Poly-L-lysine hydrobromide (PLL) working solution
    Mix 1 ml of PLL stock solution (10 mg/ml) with 9 ml of borate buffer
    Filter sterilize (0.20 µm-filter) and stored at 4 °C for up to 4 weeks
  4. Dissociation solution
    Mix 500 ml of HBSS with 3.5 ml of HEPES (1 M, pH 7.25)
    Add 5 ml penicillin/streptomycin (100x)
    Solution was freshly prepared for each experiment
  5. Glucose stock solution (30%)
    Dissolve 30 g of C6H12O6 in 50 ml of ddH2O
    Add ddH2O to 100 ml
    Filter sterilize (0.20 µm-filter) and stored at 4 °C for up to 6 months
  6. 0.5% BSA-blocking solution
    0.5 g of BSA
    Add HBSS to 100 ml
    Filter sterilize (0.20 µm-filter) and stored at 4 °C for up to 1 year
  7. DRG neuronal culture medium
    100 µl L-glutamine (200 mM)
    400 µl B-27® Supplement (50x)
    200 µl penicillin/streptomycin (100x)
    Add Neurobasal®-A medium to 20 ml
    Stored at 4 °C for up to 2 weeks
  8. DRG preparation medium
    140 µl HEPES (1 M, pH 7.25)
    200 µl glucose stock solution (30%)
    100 µl penicillin/streptomycin (100x)
    Add Minimal Essential Medium (MEM) to 10 ml
    Medium was freshly prepared for each experiment
  9. Collagenase-II solution
    Dissolve 10 mg of collagenase-II in 1 ml of dissociation solution
    Solution was freshly prepared for each experiment
  10. 2% trypsin stock solution
    Dissolve 100 mg of trypsin in 5 ml of HBSS
    Aliquot and stored at 4 °C for up to 6 months

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft to I. K. (KU 1587/4-1), C. A. H. (HU 800/6-1, RTG1715), T. H. (RTG 1715). Animal care and experimental procedures were performed in accordance with the guidelines established by the animal welfare committee of the University of Jena.

References

  1. Khaminets, A., Heinrich, T., Mari, M., Grumati, P., Huebner, A. K., Akutsu, M., Liebmann, L., Stolz, A., Nietzsche, S., Koch, N., Mauthe, M., Katona, I., Qualmann, B., Weis, J., Reggiori, F., Kurth, I., Hubner, C. A. and Dikic, I. (2015). Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522(7556): 354-358.

简介

我们在这里提供了详细的协议,用于隔离和培养的主要小鼠感觉神经元。 感觉传入假性极化神经元的细胞体位于沿着脊柱的背根神经节(DRG)中。 解离的小鼠DRG可以通过酶消化解离成单个细胞,以获得小鼠感觉神经元的原代培养物,如Khaminets等人(2015)报道的研究中所进行的。

材料和试剂

  1. T25细胞培养瓶(25cm 2)(Greiner Bio-One GmbH,目录号:690160)。
  2. 24孔板(Greiner Bio-One GmbH,目录号:662160)
  3. 封面眼镜(Marienfeld-Superior,目录号:0111550)
  4. 玻璃巴斯德移液管(Marienfeld-Superior,目录号:3233050)
  5. Filtropur S 0.2注射器过滤器(Sarstedt AG,目录号:83.1826.001)
  6. Syringe Omnifix ?(B. Braun Medical,目录号:4616103V)
  7. 具有硅胶垫的解剖皿(培养皿)(94×16mm)(Sigma-Aldrich,目录号:Z617636)
  8. Greiner培养皿(35×10mm)(Sigma-Aldrich,目录号:P5112)
  9. 15ml Cellstar离心管(Greiner Bio-One GmbH,目录号:188261)
  10. 小鼠(2至6个月)(菌株:C57BL/6)
  11. 聚-L-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P2636)
  12. HEPES(AppliChem GmbH,目录号:A3268)

  13. (Carl Roth GmbH + Co.,目录号:6943.1)
  14. 四硼酸钠(Na 2 BO 4 O 7)(Sigma-Aldrich,目录号:221732)
  15. 牛血清白蛋白(PAA,目录号:K41-001)
  16. HBSS(Thermo Fisher Scientific,Gibco TM ,目录号:14175-053)
  17. Neurobasal -A培养基(Thermo Fisher Scientific,Gibco TM ,目录号:10888022)
  18. B-27 补充(50x)(Thermo Fisher Scientific,Gibco TM ,目录号:17504-044)
  19. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco< sup> TM,目录号:25030)
  20. 青霉素/链霉素(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:15140)
  21. 最低必需培养基(MEM)(Thermo Fisher Scientific,Gibco TM ,目录号:31095-029)
  22. D(+) - 葡萄糖(C 6 H 12 S 6 O 6)(Merck Millipore Corporation,目录号:108337)
  23. 胶原酶II(Worthington Biochemical Corporation,目录号:LS004176)
  24. 马血清(Thermo Fisher Scientific,Gibco TM ,目录号:26050-88)
  25. 来自牛胰腺的胰蛋白酶(Sigma-Aldrich,目录号:T1426)
  26. β神经生长因子(β-NGF)(Preprotec,目录号:450-01)
  27. 硼酸缓冲液(参见配方)
  28. 聚-L-赖氨酸氢溴酸盐(PLL)储备溶液(见配方)
  29. 聚-L-赖氨酸氢溴酸盐(PLL)工作溶液(参见配方)
  30. 解离解决方案(参见配方)
  31. 30%葡萄糖储备溶液(见配方)
  32. 0.5%BSA封闭溶液(参见配方)
  33. DRG神经元培养基(见配方)
  34. DRG制剂(见配方)
  35. 胶原酶II溶液(参见配方)
  36. 2%胰蛋白酶原液(见配方)

设备

  1. CO 2 sub培养箱(37℃,5%CO 2浓度和95%相对湿度)(Labotect,型号:C-200)
  2. 层流工作台(Heraeus Holding)
  3. 立体显微镜(Carl Zeiss,型号:Stemi 2000C)
  4. 冷光源(配备有柔性双分支光导)(Carl Zeiss,型号:CL1500ECO)
  5. 剪刀
    1. 标准剪刀(Fine Science Tools,目录号:14002-12)
    2. 学生弹簧剪刀(Fine Science Tools,目录号:91500-09)
    3. 斜角弹簧剪刀(Fine Science Tools,目录号:15006-09)
  6. 镊子
    1. Dumont#7b医用镊子(Fine Science Tools,目录号:11270-20)
    2. Dumont#4镊子(Fine Science Tools,目录号:11242-40)
    3. Dumont#5镜面镊子(Fine Science Tools,目录号:11252-23)
  7. 本生燃烧器Fireboy plus(Integra Biosciences)
  8. 玻璃巴斯德移液管(Marienfeld-Superior,目录号:3233050)
  9. 巴斯德移液管滴管球(Thermo Fisher Scientific,目录号:03-448-25)
  10. 离心机5810R(Eppendorf AG)
  11. Pipetman P20/P200/P1000(Gilson)

程序

  1. 第1天:制备解离溶液并预冷至4℃
  2. 用聚-L-赖氨酸氢溴酸盐(PLL)包被T25细胞培养瓶。为此目的,将1ml PLL溶液(1mg/ml,在硼酸盐缓冲液中)加入到细胞培养瓶中,并在37℃温育过夜。或者,将8个盖玻片置于无菌24孔板的孔中,并在37℃下与PLL温育过夜。
  3. 第2天:吸出PLL溶液,并用无菌水漂洗细胞培养瓶三次。将烧瓶留在空气中,在层流罩中干燥15分钟
  4. 然后将烧瓶与5ml DRG神经元培养基预温育并置于培养箱中
  5. 通过向上和向下移动0.5%BSA封闭溶液三次来封闭无菌玻璃移液管。阻塞玻璃巴斯德移液管存放在层流工作台中,直到同一天使用
  6. 剪刀和镊子在70%乙醇中消毒30分钟,然后风干
  7. 通过颈脱位法杀死小鼠,并用一对标准剪刀快速解剖脊髓。为了准备脊髓,用标准剪刀在腹侧中线切开皮肤。将器官从胸腔和腹腔中取出。使用标准剪刀,两个长切口紧邻左右脊柱。尾巴和头骨被删除。最后,剩余的椎旁肌大部分被移除以暴露脊柱
  8. 使用立体显微镜小心地移除所有脊柱水平的DRG并收集在置于冰上的10-cm解剖皿中的10ml HBSS中。详细地,用学生弹簧剪刀将脊柱首先切割成三个大致相等的块,以改进随后的处理。然后,将学生弹簧剪刀的一个刀片从腹侧插入椎管中。剪刀的另一刀片在其腹侧上放置在脊柱外部。沿脊柱形成两个切口,如图1A-C和图2的方案所示,在腹侧上的中线的一个左侧和一个右侧。脊柱的切除部分被提起,脊髓从柱(参见图1D-F)。 DRG是感觉神经元的体细胞簇,并且紧密地沿着脊髓的背根定位。 DRGs位于椎间孔中并且具有长的远端和近端过程(假性极性神经元)。 DRG的特征在于圆形和??与神经纤维束的白色不同的透明外观。学生弹簧剪刀,杜蒙#7b和#4钳子用于仔细隔离三个脊髓的每个的暴露的神经节。
  9. 将分离的DRG收集在填充有5ml DRG制备培养基的35-mm培养皿中。将盘子保存在冰上。根据实验者的专业知识获得约20至40个神经节
  10. 围绕神经节的神经束的结缔组织的薄层在一对弹簧剪刀和Dumont#5镜面镊子的帮助下被去除。然后将DRG转移到在冰上填充有DRG制备培养基的第二个35-mm培养皿中。
  11. 制备胶原酶-II溶液并预热10分钟至37℃。
  12. 将分离的DRG在玻璃巴斯德吸管的帮助下在层流工作台下转移到15-ml离心管中。
  13. 在DRGs在管底部上沉降后,除去上清液并弃去。
  14. 用5ml预冷却和无菌的离解溶液冲洗DRG
  15. 再次,除去上清液。如步骤13和14所述,DRG总共洗涤三次,每次用5ml离解溶液。
  16. 在最后一次洗涤步骤后,留下2ml解离溶液并加入1ml胶原酶-II溶液
  17. DRG在37℃孵育1小时,同时每10分钟轻轻摇动隼
  18. 将2%胰蛋白酶原液的等分试样解冻并预热20分钟至37℃。
  19. 加入150μl活化胰蛋白酶
  20. 将DRG在37℃下孵育另外9分钟,同时每3分钟轻轻摇动猎鹰。
  21. 小心地除去上清液并用步骤13和14中所述的5ml离解溶液洗涤
  22. 在最后一次洗涤步骤后,留下1.5ml离解溶液
  23. 为了获得单细胞,用在步骤5中制备的玻璃巴斯德移液管解离DRG。两个玻璃巴斯德移液管被火抛光以获得较小的开口。使用具有1.1-1.3mm原始开口直径的玻璃移液管,上下移动大约10次DRG,直到溶液呈现均匀。然后使用具有较小直径的(火抛光的)巴斯德移液管,并将溶液研磨8次。最后,使用非常小直径(火抛光)的巴斯德移液管3次
  24. 将单细胞悬浮液在160×g离心5分钟。离心机的减速度减少到"3"(在10个制动斜坡中),以避免在离心机停止时小的和脆弱的细胞团分散。
  25. 从细胞沉淀中除去上清液。
  26. 将补充有10%马血清的DRG神经元培养基加入细胞沉淀;将细胞重悬浮并在160×g离心5分钟。离心机的减速度再次设置为"3"。
  27. 小心除去上清液,用1000μl移液管将细胞重悬浮于DRG神经元培养基中
  28. 将从一只小鼠获得的细胞(大约50,000至100,000个细胞)接种在一个T25细胞培养瓶中,或者在8个盖玻片(10mm直径)上,并置于37℃,5%CO 2大气层。
  29. 铺板后30分钟,用补充有β-NGF(50ng/ml)的新鲜DRG细胞培养基替换培养基。将5ml体积加入到T25烧瓶中或者对于24孔板的每个孔加入0.5ml。
  30. 原代培养物保持长达三周。每3至5天更换三分之一体积的新鲜补充了β-NGF(50ng/ml)的DRG神经元培养基。图3显示培养的DRG神经元在铺板后不同时间点的明视场显微镜图像,证明培养物中DRG神经元过程的进行性生长。神经元特异性III类β-微管蛋白的染色(参见图4)进一步说明了补充培养基的由β-NGF刺激的培养的DRG神经元的特征形态。

代表数据


图1. DRG解剖图。 A.显示脊髓的腹侧。公元前。沿脊髓的腹侧形成两个切口,一个左(B)和一个右(C)。 D-E。脊柱的切除部分被抬起。 F.从柱中除去脊髓。 G.暴露神经节并(H)从周围组织小心地分离。 I.箭头指向具有其特有的圆形,透明质外观和其近端过程的一部分的孤立的背根神经节。


图2.脊髓准备用于DRG隔离的示意图。对于脊髓管的开放,进行两个切口(红色虚线)。显示背侧的感觉神经节的位置。


图3.培养中分离的原代DRG神经元。 在体外培养3天后,(B)在第1天体外,(C)在体外培养第2天时的原代小鼠背根神经节神经元(A)和(D)在第5天在体外。比例尺:10μm


图4.荧光标记的原代DRG神经元的共焦图像 A.在培养4天后的原代小鼠背根神经节神经元。固定的细胞用神经元特异性III类β-微管蛋白染色(克隆TUJ1,Stemcell technologies,1:500)。 B.用Hoechst 33258染色细胞核。C.合并的图像。比例尺:100μm

食谱

  1. 硼酸盐缓冲液
    1.24g H sub 3 BO 3
    1.90g Na 2 B 4 SubO 4 7 / 将ddH 2 O添加到400 ml
    储存在4°C长达1年
  2. 聚-L-赖氨酸氢溴酸盐(PLL)储液
    将100mg PLL溶解在10ml ddH 2 O中 等分,并储存在-20°C长达6个月
  3. 聚-L-赖氨酸氢溴酸盐(PLL)工作溶液
    将1ml PLL储备液(10mg/ml)与9ml硼酸盐缓冲液混合 过滤除菌(0.20μm过滤器)并在4℃下储存最多4周
  4. 解离溶液
    将500ml HBSS与3.5ml HEPES(1M,pH 7.25)混合 加入5ml青霉素/链霉素(100x)
    新鲜制备用于每个实验的溶液
  5. 葡萄糖储备溶液(30%)
    将30g C 6 H 12 SO 6溶解在50ml ddH 2 O中。< br /> 将ddH 2 O加到100 ml
    过滤灭菌(0.20μm过滤器)并在4℃下贮存最多6个月
  6. 0.5%BSA封闭溶液
    0.5g BSA
    将HBSS添加至100 ml
    过滤灭菌(0.20μm过滤器),并在4°C下保存最多1年
  7. DRG神经元培养基
    100μlL-谷氨酰胺(200mM) 400μlB-27 ?补充(50x)
    200μl青霉素/链霉素(100x)
    将Neurobasal -A培养基加至20ml ml / 4℃保存2周
  8. DRG制剂培养基
    140μlHEPES(1M,pH 7.25)
    200μl葡萄糖储备溶液(30%)
    100μl青霉素/链霉素(100x)
    将最小必需培养基(MEM)加至10ml ml / 新鲜制备用于每次实验的培养基
  9. 胶原酶II溶液
    将10 mg胶原酶-II溶解在1 ml离解溶液中
    新鲜制备用于每个实验的溶液
  10. 2%胰蛋白酶原液
    将100mg胰蛋白酶溶解在5ml HBSS中 等分,并在4°C下存储最多6个月

致谢

这项工作得到Deutsche Forschungsgemeinschaft对I.K.(KU 1587/4-1),C.A.H。(HU 800/6-1,RTG1715),T.H。(RTG 1715)的赠款的支持。根据由耶拿大学动物福利委员会制定的指南进行动物护理和实验程序。

参考文献

  1. Khaminets,A.,Heinrich,T.,Mari,M.,Grumati,P.,Huebner,AK,Akutsu,M.,Liebmann,L.,Stolz,A.,Nietzsche,S.,Koch,N.,Mauthe M.,Katona,I.,Qualmann,B.,Weis,J.,Reggiori,F.,Kurth,I.,Hubner,CA和Dikic,I。(2015)。 通过选择性自噬调节内质网周转。 自然 522(7556):354-358。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Heinrich, T., Hübner, C. A. and Kurth, I. (2016). Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons. Bio-protocol 6(7): e1785. DOI: 10.21769/BioProtoc.1785.
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