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Mouse Subependymal Zone Explants Cultured on Primary Astrocytes
小鼠室管膜下区外植体在原代星形胶质细胞中的培养   

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Abstract

Neuroblast migration is a major component in the process of neuronal circuit assembly. In the rodent postnatal brain, the subependymal zone (SEZ) is the largest neurogenic niche where new neurons are born throughout life. These cells migrate several millimeters following a specific pathway called the rostral migratory stream (RMS) towards the olfactory bulb. Once they reach their final destination, they differentiate mainly as interneurons and integrate into already established neuronal circuits. Along the RMS, neuroblasts use a particular mode of migration known as chain migration. They stay attached to each other while migrating and are intimately associated with adjacent astrocytic processes. To dissect the molecular and cellular signals involved in neuroblast migration, we developed an in vitro system consisting of SEZ-derived explants co-cultured on top of an astrocyte monolayer.

Keywords: Neuroblast(成神经细胞), Neurogenesis(神经发生), Migration(迁移), RMS(RMS), Postnatal(产后)

Materials and Reagents

  1. 10 cm Petri dishes (Greiner Bio-One GmbH, catalog number: 664160 )
  2. 15-ml tube
  3. 75 cm2 plastic flasks (Sigma-Aldrich, Corning® CellBIND®, catalog number: CLS3290 )
  4. Glass coverslips (Marienfeld-superior 18 mm Φ)
  5. 24-well plates (Sigma-Aldrich, Greiner Cellstar®, catalog number: M8812-100EA )
  6. Breakable razor blades (Fine Science Tools, catalog number: 10050-00 )
  7. Puradisc 30 syringe filters (Whatman, Whatman®, catalog number: 10462200 )
  8. Animals: Wild-type C57BL/6 mice (Charles River)
    Note: Animal care and procedures were according to local and international regulations for the use of experimental animals. Animal handling was performed by trained staff certified by the Federation of European Laboratory Animal Science Associations (FELASA).
  9. Antibodies:
    1. Goat anti-DCX (Santa Cruz Biotechnology, catalog number: sc-8066 )
    2. Mouse anti-GFAP (Sigma-Aldrich, catalog number: G-3893 )
    3. Anti-goat conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, catalog number: A11055 )
    4. Anti-mouse conjugated to Alexa Fluor 647 (Thermo Fisher Scientific, catalog number: A31571 )
  10. Papain (Sigma-Aldrich, catalog number: P4762 )
  11. DNase I (Sigma-Aldrich, catalog number: D4513 )
  12. Dulbecco’s modified Eagle’s media (DMEM) (Thermo Fisher Scientific, catalog number: 41965039 )
  13. Fetal bovine serum (FBS), heat inactivated (Thermo Fisher Scientific, catalog number: 10270-106 )
  14. Trypsin–EDTA (0.05%) (Thermo Fisher Scientific, catalog number: 25300-054 )
  15. Poly-L-lysine (Sigma-Aldrich, catalog number: P9155 )
  16. Neurobasal media (Thermo Fisher Scientific, 21103049 )
  17. 4% paraformaldehyde (PFA) (Carl Roth, catalog number: P087.3 )
  18. Phosphate buffer saline (PBS)
  19. PBS-Triton
  20. Bovine Serum Albumin (BSA)
  21. Mowiol (Sigma-Aldrich, catalog number: 324590 )
  22. NaCl (Sigma-Aldrich, catalog number: 31434-1KG-R )
  23. KCl (AppliChem GmbH, catalog number: A1039 )
  24. Na2HPO4 (VWR International, Prolabo, catalog number: 28029.292 )
  25. KH2PO4 (GERBU Biotechnik, catalog number: 2018 )
  26. Glucose (Sigma-Aldrich, catalog number: G-7021 )
  27. HEPES (Carl Roth Gmbh, catalog number: 9105.2 )
  28. B27 supplement (Thermo Fisher Scientific, 17504044 )
  29. L-glutamine (Thermo Fisher Scientific, catalog number: 25030-024 )
  30. Pen/Strep (Thermo Fisher Scientific, catalog number: 15140-122 )
  31. PBS-Glucose-HEPES (see Recipes)
  32. Neurobasal complete media (see Recipes)
  33. Dulbecco’s modified Eagle’s medium (DMEM)-10% serum (see Recipes)
  34. Papain solution (see Recipes)
  35. DNase I solution (see Recipes)

Equipment

  1. Standard pattern scissors sharp/blunt 14,5cm (Fine Science Tools, catalog number: 14101-14 )
  2. Stereomicroscope (Carl Zeiss, model: STEMI SV 6 ) supplemented with a halogen light source (Schott KL, model: 1500 LCD )
  3. Horizontal laminar flow bench (Azbil Telstar, model: H-100 )
  4. Biological safety cabinet (The Baker Company, model: SterilGARD e3 )
  5. Dumont #5 Forceps (Fine Science Tools, catalog number: 11251-20 )
  6. Graefe forceps-straight/serrated (Fine Science Tools, catalog number: 11050-10 )
  7. Neubauer chamber
  8. Scalpel Handle #3-12 cm (Fine Science Tools, catalog number: 10003-12 )
  9. Scalpel Blades #15 (Fine Science Tools, catalog number: 10015-00 )
  10. 37 °C, 5% CO2 incubator (Labotect, model: C200 )
  11. Bright field microscope
  12. Confocal microscope (Carl Zeiss, model: LSM 700 )

Procedure

An overview of the whole procedure is schematized in Figure 1.


Figure 1. Summary of the complete procedure. Scheme of the steps described in the protocol, numbered as in the text (Procedure section).

  1. Astrocyte monolayer preparation from cortical tissue
    We use a modified version of the protocol previously described by Banker and Goslin (1991), as follows:
    1. Sacrifice P4-7 old mice by decapitation with sharp scissors. Remove the brains and transfer them to a sterile Petri dish with cold PBS-glucose-HEPES buffer under a stereomicroscope placed in a horizontal laminar flow bench.
    2. Remove the meninges with a fine forceps and dissect the cortex, mince it with a scalpel into small pieces of approx. 2 mm of diameter and transfer them to a 15-ml tube with PBS-glucose on ice. We use one tube per ~100-200 μl of tissue, normally we prepare 4 tubes in parallel and the cortex from one mouse is enough to obtain the material.
    3. Prepare papain (0.08%)/DNase I (0.001%) (final concentration) in 1 ml of PBS-glucose, add this solution to the tissues and incubate them for 3 min at 37 °C. Immediately pipette out the solution leaving the tissues on the bottom of the tube and wash them twice by adding 1 ml of DMEM-10% serum media at RT, decanting the tissue and removing the solution each time.
    4. Resuspend the tissue in DMEM-10% serum and to obtain a single cell suspension gently pipette up and down approx. 20 times with a 200-μl tip until the solution appears homogeneous.
      Note: In case some tissue pieces remain undigested, discard them from the cell suspension without further pipetting to avoid damaging the cells.
    5. Count the cells in a Neubauer chamber and plate them in 75 cm2 plastic flasks at a density of 100,000 cells/ml in DMEM-10% serum. We plate 2 x 106 cells per flask (20 ml) and keep the cells in culture for at least one week before splitting. To split the cells remove the media and add 2 ml trypsin solution per flask. Incubate the flasks for 5 min at 37 °C and subsequently inactivate the trypsin with 10 ml DMEM-10% FBS. Detach the cells from the flask by stringently pipetting the media and pellet down by centrifugation for 5 min at 800 x g. Discard the media and replace it with 10 ml fresh DMEM-10% FBS.
    6. One day prior explant plating, re-plate the astrocytes on glass coverslips coated with poly-lysine (in a 24-well plate). Coating is performed by adding 200 µl of poly-lysine solution on top of each coverslip placed inside a 24-well plate and incubating at 37 °C for 2-3 h. Afterwards the poly-lysine solution should be removed and the wells should be washed 3 times with 1 ml sterile PBS. Plate the astrocytes on the coverslips at a density of 35,000 cells/well in a volume of 150 µl avoiding that the solution spills out of the glass.  Wait until the cells attach (approx. 15 min) and add 350 µl of media to each well.
      Note: The astrocyte monolayer should be confluent at the time of plating the explants because neuroblasts migrate avoiding the empty surfaces.
    7. Manipulation of gene expression in astrocytes via AAV-viral infections
      Specific gene overexpression or knockdown can be achieved by infecting the cells with recombinant adeno-associated viral (AAV) vectors. The viral backbone contains the sequence of the gene of interest under a general promoter (overexpression) or a shRNA sequence driven by the H1 or U6 promoter (knockdown). In addition, a fluorescent marker is expressed to identify the infected cells.
      1. AAVs are generated with the packaging cell line HEK 293 transfected with the plasmid for the viral backbone and the helper plasmids pDP1 and pDP2. The viral particles are purified as previously described (Alfonso et al., 2015). A detailed protocol can be found in McClure et al., 2011.
      2. Infect 70% confluent astrocyte cultures growing in 10 cm plates with the virus by adding the viral solution in the media (1:10 final dilution, the titer of the virus was approx. 106 cfu/µl). Replace the media on the following day and keep the cells in culture for at least 6 days before re-plating in 24-well plates for explant plating. Normally, between 60-90% of the cells are infected with the virus. Alternatively, primary astrocytes can also be infected with lentivirus. Other methods like lipid-mediated transfection or electroporation can also be used to express an exogenous sequence although they usually have a lower efficiency.
  2. SEZ dissection and explant plating
    1. Sacrifice P4-10 old mice by decapitation and remove their brains in cold PBS-glucose-HEPES buffer as before. Cut coronal slices of approx. 400 μm at the level of the lateral ventricles (2 mm posterior to the end of the olfactory bulb) with a breakable scalpel blade and select the sections containing the SEZ (Figure 2A and 2B).
    2. Dissect out the wall of the ventricle with a scalpel (Figure 2C-E) and place it in a clean 10 cm Petri dish with PBS-glucose-HEPES on ice.
      Note: This cut should be as close to the ventricle as possible avoiding that the dissected area contains parts of the striatum. 
    3. Mince the tissue in small pieces of ~200-400 μm diameter with a scalpel (Figure 2F), and either incubate them with an AAV-virus before plating (see step 2d), or immediately plate them on top of the primary astrocyte cultures prior replacement of the media with Neurobasal complete media. Plate 2-3 explants (distant from each other) per well (24-well plate) with a pipette (20 μl tip) and carefully transfer the plate to the incubator avoiding shifting the tissues.
      Note: The explants require approx. 4-6 h to attach to the surface, hence it is important to keep the plates in the incubator avoiding any handling during this time.


      Figure 2. Dissection of SEZ-derived explants. A. A brain from a P6-old mouse was placed on a Petri dish and a coronal slice at the level of the lateral ventricles (indicated with the black dotted line) was dissected with a breakable scalpel blade. B. The brain section obtained in (A) was cut with a scalpel along the dotted line and the wall of the ventricle exposed (C) in order to allow its dissection from the brain slice (D and E). F. The dissected tissue was further cut into small pieces, and was then ready to be plated on the coverslips containing the astrocyte cultures. Cx: cortex, LV: lateral ventricle, St: striatum.

    4. Manipulation of gene expression in neuroblasts via AAV-viral infections
      1. AAVs are generated as above (step 1g)
      2. After the SEZ-explant dissection, place the small pieces of tissue (30-40 explants) in a Petri dish and cover them with ~20 μl of purified virus (106 cfu/µl) for 15-20 min at room temperature. Dilute the viral solution by adding 200 µl of PBS-glucose and plate the infected tissue as described above (step 2c). Check for positive infection with an inverted fluorescent microscope 2 days after plating. Normally, approx. 70% of the cells are infected with the virus.
  3. Neuroblasts visualization
    One day after plating, the explants are attached to the substrate and the neuroblasts migrating out of the tissue can be observed under a bright field microscope.
    1. Immunostaining: one to three days after plating, fix the explants by incubating the coverslips in 4% PFA (in PBS) for 30 min at room temperature, permeabilize in 1% PBS-triton for 15 min at RT, block with 3% PBS-BSA for at least 30 min at RT and incubate with goat anti-DCX (neuroblast marker, 1:500) and mouse anti-GFAP (astrocyte marker, 1:1,000) antibodies in 3% PBS-BSA overnight at 4 °C. Next day, wash the coverslips 3 times with PBS, incubate them with the secondary antibodies anti-goat conjugated to Alexa Fluor 488 (1:1,000) and anti-mouse conjugated to Alexa Fluor 647 (1:1,000) for 1 h at RT, wash again 3 times with PBS and mount in Mowiol mounting medium.
    2. Image acquisition: the immunostainings can be imaged with a confocal microscope using a 20x objective and the tile-scan function (see examples in Figure 3).


      Figure 3. SEZ-derived explants on astrocytes cultures. Coverslips homogenously (A) or irregularly (B) covered by astrocytes were used as a substrate for SEZ-derived explants. Two days after plating, the cells were stained with antibodies against the astrocyte marker GFAP and the neuroblast marker DCX, as indicated in the pictures. Note that neuroblasts do not migrate over areas devoid of astrocytes. Scale bars: 100 μm.

Representative data

Different parameters can be measured once the neuroblasts have migrated out of the explant:

  1. Mode of migration: we measured whether the cells migrated as single cells or contacting each other since cell-cell adherence among neuroblasts is important to maintain the integrity of the chains during chain migration (Figure 4A and 4B).
  2. Efficiency of migration: we measured the distance migrated by the neuroblasts from the explant (Figure 4A and 4C).


    Figure 4. Cell-cell adherence and distance migrated by neuroblasts. A. Picture of an explant one day after plating stained with anti-DCX antibodies. Blue lines show examples of the distance measured from the explant for two neuroblasts (d1 and d2). The inset is an enlargement of the boxed area showing an example of a neuroblast migrating as single cell (s) and another one associated with other neuroblasts (a). B. Quantification of the percentage of cells migrating as single cells for 13 explants. C. Quantification of the distance covered by the neuroblasts in one explant. Scale bars: 50 μm.

Notes

  1. In our hands, the reproducibility of the technique is high as long as the dissection is precise. It is crucial to dissect only tissue from the wall of the ventricle to prepare the explants since contaminating tissue from adjacent brain areas does not contain neuroblasts. In a standard experiment plating 3 explants per well in a 24 well plate, we expect to obtain migrating neuroblasts in at least 20 wells.

Recipes

  1. PBS-Glucose-HEPES
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    6 g glucose
    7.38 g HEPES
    Dissolve in double-distilled water (1 L final volume), adjust pH at 7.38 and sterilize by autoclaving.
    The solution can be stored at 4 °C for up to 8 weeks.
  2. Neurobasal complete media
    483.75 ml Neurobasal Media
    10 ml B27 supplement
    1.25 ml L-glutamine
    5 ml Pen/Strep
    Solution must be prepared sterile.
    The solution can be stored at 4 °C for up to 6 weeks.
  3. Dulbecco’s modified Eagle’s medium (DMEM)-10% serum
    443.75 ml DMEM
    50 ml FBS (heat inactivated)
    1.25 ml L-glutamine
    5 ml Pen/Strep
    Solution must be prepared sterile.
    The solution can be stored at 4 °C for up to 6 weeks.
  4. Papain solution
    100.2 mg Papain
    20 mg cysteine
    20 mg EDTA
    To be dissolved in 60 ml PBS and filter sterilized using a 0.2 µm Filter.
    The solution can be stored at -20 °C for up to 6 months.
  5. DNase I solution
    14.2 mg solid DNase I
    To be dissolved in 14.2 ml bidistilled water and filter sterilized using a 0.2 µm Filter.
    The solution can be stored at -20 °C for up to 6 months.

Acknowledgments

This protocol was successfully used and described in a shorter version in the published studies Alfonso et al., 2015 and Le Magueresse et al., 2012. We thank J. Friemann and R. Hinz-Herkommer for excellent technical assistance. This work was supported by the German Federal Ministry for Education and Research (Grant BMBF 01GQ1405 to J.A.).

References

  1. Alfonso, J., Penkert, H., Duman, C., Zuccotti, A. and Monyer, H. (2015). Downregulation of Sphingosine 1-Phosphate Receptor 1 Promotes the switch from tangential to radial migration in the OB. Journal of Neuroscience 35(40): 13659-13672.
  2. Banker, G. and Goslin, K. (1991). Culturing nerve cells. Cambridge (MA): MIT Press.
  3. Le, Magueresse, C., Alfonso, J., Bark, C., Eliava, M., Khrulev, S. and Monyer, H. (2012). Subventricular zone-derived neuroblasts use vasculature as a scaffold to migrate radially to the cortex in neonatal mice. Cerebral Cortex 22(10): 2285-2296.
  4. McClure, C., Cole, K. L., Wulff P., Klugmann, M. and Murray, A. J. (2011). Production and titering of recombinant adeno-associated viral vectors. J Vis Exp 27:e3348.

简介

神经母细胞迁移是神经元电路组装过程中的主要组成部分。 在啮齿动物出生后的大脑中,子宫颈区(SEZ)是新生神经元生命中最大的神经源性生态位。 这些细胞沿着称为传播流(RMS)的特定途径朝向嗅球迁移几毫米。 一旦达到其最终目的地,它们主要区分为中间神经元并整合到已建立的神经元回路中。 沿着RMS,神经母细胞使用称为链转移的特定迁移模式。 它们在迁移时保持相互依赖,并与相邻的星形细胞过程密切相关。 为了解剖神经母细胞迁移中涉及的分子和细胞信号,我们开发了一种体外系统,其由在星形胶质细胞单层顶部共培养的经SEZ衍生的外植体组成。

关键字:成神经细胞, 神经发生, 迁移, RMS, 产后

材料和试剂

  1. 10cm培养皿(Greiner Bio-One GmbH,目录号:664160)
  2. 15毫升管
  3. 75cm 2塑料烧瓶(Sigma-Aldrich,Corning CellBIND ,目录号:CLS3290)中。
  4. 玻璃盖片(Marienfeld-superior18mmΦ)
  5. 24孔板(Sigma-Aldrich,Greiner Cellstar ,目录号:M8812-100EA)
  6. 可破碎的剃刀刀片(Fine Science Tools,目录号:10050-00)
  7. Puradisk 30注射器过滤器(Whatman,Whatman ,目录号:10462200)
  8. 动物:野生型C57BL/6小鼠(Charles River)
    注意:动物护理和程序根据当地和国际规定使用实验动物。动物处理由经欧洲实验动物科学协会联合会(FELASA)认证的受过训练的工作人员进行。
  9. 抗体:
    1. 山羊抗DCX(Santa Cruz Biotechnology,目录号:sc-8066)
    2. 小鼠抗GFAP(Sigma-Aldrich,目录号:G-3893)
    3. 与Alexa Fluor 488偶联的抗山羊(Thermo Fisher Scientific,目录号:A11055)
    4. 与Alexa Fluor 647(Thermo Fisher Scientific,目录号:A31571)缀合的抗小鼠
  10. 木瓜蛋白酶(Sigma-Aldrich,目录号:P4762)
  11. DNase I(Sigma-Aldrich,目录号:D4513)
  12. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,目录号:41965039)
  13. 胎牛血清(FBS),热灭活(Thermo Fisher Scientific,目录号:10270-106)
  14. 胰蛋白酶-EDTA(0.05%)(Thermo Fisher Scientific,目录号:25300-054)
  15. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P9155)
  16. Neurobasal培养基(Thermo Fisher Scientific,21103049)
  17. 4%多聚甲醛(PFA)(Carl Roth,目录号:P087.3)
  18. 磷酸盐缓冲液(PBS)
  19. PBS-Triton
  20. 牛血清白蛋白(BSA)
  21. Mowiol(Sigma-Aldrich,目录号:324590)
  22. NaCl(Sigma-Aldrich,目录号:31434-1KG-R)
  23. KCl(AppliChem GmbH,目录号:A1039)

  24. (VWR International,Prolabo,目录号:28029.292)。

  25. (GERBU Biotechnik,目录号:2018)。
  26. 葡萄糖(Sigma-Aldrich,目录号:G-7021)
  27. HEPES(Carl Roth Gmbh,目录号:9105.2)
  28. B27补充(Thermo Fisher Scientific,17504044)
  29. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030-024)
  30. Pen/Strep(Thermo Fisher Scientific,目录号:15140-122)
  31. PBS-Glucose-HEPES(参见配方)
  32. 神经基础完全培养基(见配方)
  33. Dulbecco's改良的Eagle培养基(DMEM)-10%血清(参见Recipes)
  34. 木瓜蛋白酶溶液(参见配方)
  35. DNase I解决方案(参见配方)

设备

  1. 标准图案剪刀尖/钝14,5厘米(Fine Science Tools,目录号:14101-14)
  2. 补充有卤素光源(Schott KL,型号:1500LCD)的立体显微镜(Carl Zeiss,型号:STEMI SV 6)
  3. 水平层流台(Azbil Telstar,型号:H-100)
  4. 生物安全柜(贝克公司,型号:SterilGARD e3)
  5. Dumont#5镊子(Fine Science Tools,目录号:11251-20)
  6. Graefe镊子 - 直/锯齿(Fine Science Tools,目录号:11050-10)
  7. Neubauer房间
  8. 手术刀柄#3-12厘米(Fine Science Tools,目录号:10003-12)
  9. Scalpel Blades#15(Fine Science Tools,目录号:10015-00)
  10. 37℃,5%CO 2培养箱(Labotect,型号:C200)中。
  11. 明场显微镜
  12. 共聚焦显微镜(Carl Zeiss,型号:LSM 700)

程序

整个过程的概述如图1所示。

图1.完整过程的摘要。协议中描述的步骤的计划,编号为文本(过程部分)。

  1. 皮质组织的星形胶质细胞单层制剂
    我们使用以前由Banker和Goslin(1991)描述的协议的修改版本,如下:
    1. 用锋利的剪刀断头处死P4-7老鼠。取出大脑,并将其转移到无菌陪替氏培养皿与冷PBS-葡萄糖HEPES缓冲液在立体显微镜下放置在水平层流台。
    2. 用细镊子去除脑膜,解剖大脑皮层,用手术刀将其切成小块大约。直径为2mm,并将其转移到冰上含有PBS-葡萄糖的15-ml试管中。我们使用一个管?100-200微升的组织,通常我们准备4管并行,一个小鼠的皮质足以获得材料。
    3. 在1ml PBS-葡萄糖中制备木瓜蛋白酶(0.08%)/DNA酶I(0.001%)(终浓度),将该溶液加入组织中并在37℃下孵育3分钟。立即移出溶液,留下管底部的组织,并通过在室温下加入1ml DMEM-10%血清培养基洗涤两次,倾析组织并每次除去溶液。
    4. 重悬在DMEM-10%血清中的组织,并获得单细胞悬液轻轻吸上下大约。 20倍用200-μl提示,直到溶液显示均匀。
      注意:如果一些组织片未被消化,则将其从细胞悬浮液中丢弃,而不用进一步移液以避免损伤细胞。
    5. 在Neubauer室中计数细胞,并将它们以100,000细胞/ml的密度在75cm 2的 2 在DMEM-10%血清中。我们在每个烧瓶(20ml)中铺平板2×10 6 个细胞,并将细胞培养至少一周分割前。为了分裂细胞,除去培养基并且每个烧瓶中加入2ml胰蛋白酶溶液。在37℃下孵育5分钟,随后用10ml DMEM-10%FBS灭活胰蛋白酶。通过严格吸取培养基并且以800×g离心5分钟使沉淀物从烧瓶中分离细胞。弃去培养基并更换为10ml新鲜DMEM-10%FBS。
    6. 在外植体铺板前一天,将星形胶质细胞重新铺在涂覆有聚赖氨酸的玻璃盖玻片上(在24孔板中)。通过在放置在24孔板内的每个盖玻片的顶部上加入200μl聚赖氨酸溶液并在37℃孵育2-3小时来进行包被。之后,应去除聚赖氨酸溶液,并用1ml无菌PBS洗涤孔3次。将盖玻片上的星形胶质细胞以35,000个细胞/孔的密度铺在150μl的体积中,避免溶液溢出玻璃杯。等待细胞附着(约15分钟),并添加350微升培养基到每个孔。
      注意:星形胶质细胞单层在铺板外植体时应该是融合的,因为成神经细胞迁移避免了空表面。
    7. 通过AAV病毒感染操作星形胶质细胞中的基因表达
      特异性基因过表达或敲低可以通过用重组腺相关病毒(AAV)载体感染细胞来实现。病毒骨架含有在一般启动子(过表达)下的感兴趣的基因的序列或由H1或U6启动子(敲低)驱动的shRNA序列。此外,表达荧光标记以鉴定受感染的细胞。
      1. AAV是用用于病毒骨架的质粒和辅助质粒pDP1和pDP2转染的包装细胞系HEK 293产生的。如前所述纯化病毒颗粒(Alfonso等人,2015)。详细的方案可以在McClure等人,2011中找到。
      2. 通过在培养基中加入病毒溶液(1:10最终稀释,病毒的滴度为约10 6 cfu /μl)感染在具有病毒的10cm平板中生长的70%汇合星形细胞培养物。 。更换第二天的培养基,并保持细胞培养至少6天,然后重新电镀在24孔板的外植体电镀。通常,60-90%的细胞被病毒感染。或者,原代星形胶质细胞也可以被慢病毒感染。其它方法如脂质介导的转染或电穿孔也可用于表达外源序列,尽管它们通常具有较低的效率。
  2. SEZ解剖和移植电镀
    1. 牺牲P4-10老鼠,通过断头,并如前所述在冷PBS-葡萄糖-HEPES缓冲液中除去其大脑。切割大约的冠状切片。用可破裂的手术刀刀片在侧脑室(在嗅球端部后面2mm)的水平处400μm,并选择含有SEZ的切片(图2A和2B)。
    2. 用解剖刀(图2C-E)解剖心室的壁,并将其放在一个干净的10厘米培养皿与PBS-葡萄糖-HEPES冰上。
      注意:这切口应尽可能靠近心室,避免解剖区域包含纹状体的部分。
    3. 用电刀(图2F)将组织切成?200-400μm直径的小块,并在铺板前用AAV病毒孵育(见步骤2d),或立即将它们铺在原代星形细胞培养物的顶部用Neurobasal完全培养基替换培养基。用移液管(20μl吸头)每孔(每个孔2-3个外植体(远离彼此))(24孔板),并小心地将板转移到培养箱中,避免移动组织。
      注意:外植体需要约。 4-6小时以附着到表面,因此重要的是保持培养板中的板避免在此期间的任何处理。


      图2.来自SEZ的外植体的解剖A.将来自P6老鼠的脑置于培养皿上,并在侧脑室水平处显示冠状切片(表示为与黑色虚线)用可断裂的手术刀刀片切开。 B.(A)中获得的脑切片用手术刀沿着虚线切开,并且使心室壁暴露(C),以允许其从脑切片(D和E)中解剖。 F.将切开的组织进一步切成小块,然后准备接种在含有星形胶质细胞培养物的盖玻片上。 Cx:皮质,LV:侧脑室,St:纹状体
    4. 通过AAV病毒感染在神经母细胞中操纵基因表达
      1. 如上产生AAV(步骤1g)
      2. 在SEZ-外植体解剖之后,将小片组织(30-40外植体)置于培养皿中并用?20μl纯化的病毒(10 6 cfu /μl)覆盖它们15-在室温下20分钟。通过加入200μlPBS-葡萄糖稀释病毒溶液,并如上所述平板感染的组织(步骤2c)。平板2天后,用倒置荧光显微镜检查阳性感染。通常, 70%的细胞被病毒感染
  3. 神经细胞可视化
    在接种后一天,将外植体附着于基底,并且可以在明视野显微镜下观察迁移出组织的成神经细胞。
    1. 免疫染色:铺板后1-3天,通过将盖玻片在4%PFA(在PBS中)在室温下孵育30分钟来固定外植体,在室温下在1%PBS-triton中透化15分钟,用3%PBS- BSA在室温下孵育至少30分钟,并且在4℃下在3%PBS-BSA中与山羊抗DCX(成神经细胞标记,1:500)和小鼠抗GFAP(星形胶质细胞标记,1:1000)抗体孵育过夜。第二天,用PBS洗涤盖玻片3次,将其与缀合于Alexa Fluor 488(1:1,000)的第二抗体抗山羊和与Alexa Fluor 647(1:1,000)缀合的抗小鼠在室温孵育1小时,再次用PBS清洗3次,并安装在Mowiol安装介质中。
    2. 图像采集:免疫染色可以使用共聚焦显微镜使用20倍物镜和平铺扫描功能(参见图3中的示例)成像。


      图3.星形胶质细胞培养物上的SEZ衍生的外植体。将均匀地(A)或不规则地(B)由星形胶质细胞覆盖的盖玻片用作SEZ衍生的外植体的底物。平板两天后,用针对星形胶质细胞标记物GFAP和成神经细胞标记物DCX的抗体对细胞染色,如图所示。注意,成神经细胞不在没有星形胶质细胞的区域上迁移。比例尺:100μm。

代表数据

一旦神经母细胞迁移出外植体,可以测量不同的参数:

  1. 迁移模式:我们测量细胞是迁移为单个细胞还是彼此接触,因为成神经细胞中的细胞 - 细胞粘附对于在链迁移期间保持链的完整性是重要的(图4A和4B)。
  2. 迁移效率:我们测量了由外植体形成的成神经细胞迁移的距离(图4A和4C)。


    图4.细胞 - 细胞粘附和由成纤维细胞迁移的距离。 A.用抗DCX抗体染色后一天的外植体的图片。蓝线显示从外植体测量的两种成神经细胞(d1和d2)的距离的实例。插图是加框区域的放大图,显示了作为单细胞迁移的成神经细胞和与其他成神经细胞(a)相关的另一个的示例。 B.定量作为13个外植体的单细胞迁移的细胞的百分比。 C.在一个外植体中由成神经细胞覆盖的距离的定量。比例尺:50μm

笔记

  1. 在我们的手中,该技术的再现性高,只要解剖是精确的。至关重要的是从心室壁仅切下组织以制备外植体,因为来自相邻脑区域的污染组织不含有成神经细胞。在标准实验中,在24孔板中每孔接种3个外植体,我们预期在至少20个孔中获得迁移的成神经细胞。

食谱

  1. PBS-Glucose-HEPES
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
    7.38g HEPES
    溶解在双蒸水(1L终体积)中,调节pH在7.38并通过高压灭菌灭菌。
    溶液可在4℃下储存最多8周。
  2. 神经基础完全培养基
    483.75 ml Neurobasal Media
    10ml B27补充剂
    1.25ml L-谷氨酰胺 5ml Pen/Strep
    溶液必须无菌制备 溶液可在4℃下储存最多6周
  3. Dulbecco改良的Eagle培养基(DMEM)-10%血清 443.75ml DMEM
    50ml FBS(热灭活) 1.25ml L-谷氨酰胺 5ml Pen/Strep
    溶液必须无菌制备 溶液可在4℃下储存最多6周
  4. 木瓜溶液
    100.2mg木瓜蛋白酶
    20mg半胱氨酸
    20mg EDTA
    溶解于60ml PBS中,并使用0.2μm过滤器过滤灭菌 溶液可以在-20°C下储存长达6个月。
  5. DNase I溶液
    14.2mg固体DNA酶I
    将其溶解在14.2ml双蒸馏水中,并使用0.2μm过滤器过滤灭菌 该溶液可在-20°C下储存长达6个月。

致谢

该方案在已发表的研究中被成功地使用并描述在较短的版本中。2015年和Le Magueresse等人,2012年。我们感谢J. Friemann和R. Hinz-Herkommer提供优秀的技术援助。这项工作得到德国联邦教育和研究部(Grant BMBF 01GQ1405 to J.A.)的支持。

参考文献

  1. Alfonso,J.,Penkert,H.,Duman,C.,Zuccotti,A.和Monyer,H.(2015)。  鞘氨醇1-磷酸受体1的下调促进OB中从切向转向径向迁移。 35(40):13659-13672。
  2. Banker,G。和Goslin,K。(1991)。  培养神经细胞。 剑桥 (MA):MIT新闻。
  3. Le,Magueresse,C.,Alfonso,J.,Bark,C.,Eliava,M.,Khrulev,S。和Monyer,H。(2012)。  室下区衍生的成神经细胞使用脉管系统作为支架在新生小鼠中径向迁移到皮层。 Cerebral Cortex 22(10):2285-2296。
  4. McClure,C.,Cole,KL,Wulff P.,Klugmann,M和Murray,AJ(2011)。  重组腺相关病毒载体的生产和滴定。


    27:e3348。
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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: © 2016 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. Dumitru, I., Monyer, H. and Alfonso, J. (2016). Mouse Subependymal Zone Explants Cultured on Primary Astrocytes . Bio-protocol 6(14): e1876. DOI: 10.21769/BioProtoc.1876.
  2. Alfonso, J., Penkert, H., Duman, C., Zuccotti, A. and Monyer, H. (2015). Downregulation of Sphingosine 1-Phosphate Receptor 1 Promotes the switch from tangential to radial migration in the OB. Journal of Neuroscience 35(40): 13659-13672.
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