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Dye Labeling of Live Nippostrongylus brasiliensis Larvae for Visualization in Host Tissue
对活体巴西日圆线虫幼虫进行有别于宿主的染色标记   

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Abstract

Visualization of the interaction between parasitic nematodes and their host enables a better understanding of the development of the nematode during the infectious stages of its life cycle and of the effects of host response on nematode integrity in tissues. Appropriate live imaging of these nematode/host interactions, to date has been hindered by the lack of appropriate molecular tools or efficient labeling agents. Here, we present techniques for the live labeling of the nematode parasite Nippostrongylus brasiliensis (N. brasiliensis) that allows visualization of the parasite in the mouse host for up to 24 h. The external sheath can be labeled with CFSE allowing infective larvae to be identified and followed until the stage of exsheathment. The internal labeling of infective parasites can be performed by ingestion of NY microspheres. The worms can continue to be identified for up to 24 h following exsheathment. This should be applicable to other parasitic nematodes.

Keywords: Nippostrongylus brasiliensis(巴西钩虫), Nematode(线虫), Hookworm(钩虫), Intravital imaging(活体成像), Microscopy(显微镜)

Materials and Reagents

  1. 200 μl pipette tips (Corning, Axygen®, catalog number: T-300-L-R-S )
  2. 10 μl pipette tips (Corning, Axygen®, catalog number: T-300-R-S )
  3. 1 ml pipette tips (Corning, Axygen®, catalog number: T-1000-C-L-R-S )
  4. 1.7 ml MaxyClear Snaplock Microcentrifuge Tube (Corning, Axygen®, catalog number: MCT-175-C )
  5. 24-well Tissue culture plate (Thermo Fisher Scientific, FalconTM, catalog number: 353047/08-772-1 )
  6. Insulin syringe 29 G, 0.3 ml ultrafine (BD, catalog number: 320431 )
  7. 15 ml Conical Centrifuge Tubes (Thermo Fisher Scientific, FalconTM, catalog number: 352096 / 14-959-49B )
  8. Microscope slides (Thermo Fisher Scientific, catalog number: LBS2951RC )
  9. Petri dish, 60 mm x 15 mm (In Vitro Technologies, catalog number: FAL351007 )
  10. Tin-foil (any grocery store)
  11. N. brasiliensis L3 infective larvae (iL3) (Camberis et al., 2003)
    Note: Camberis et al. (2003) provides a detailed review on the preparation of N. brasiliensis.
    Note: There are several labs that maintain the life cycle and can be contacted through their publication address.
  12. C57Bl/6J Mouse (or strain of choice)
  13. Fluoresbrite® YO Carboxylate Microspheres 0.50 µm (Polysciences, catalog number: 18720 )
  14. 5(6)-CFDA, SE; CFSE (5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester), mixed isomers (Life Technologies, Molecular ProbesTM, catalog number: C1157 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C1157”.
  15. DMEM, high glucose, pyruvate (Life Technologies, GibcoTM, catalog number: 11995-065 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065”.
  16. PBS (DPBS, no calcium, no magnesium) (Life Technologies, GibcoTM, catalog number: 14190-144 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144”.
  17. Penicillin/Streptomicin 10,000 U/ml (Life Technologies, GibcoTM, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  18. Gentamicin solution 10 mg/ml (Sigma-Aldrich, catalog number: G1272 )
  19. Fetal Calf Serum, qualified, US origin (Life Technologies, GibcoTM, catalog number: 26140-079 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 26140-079”.
  20. TWEEN® 20 (Sigma-Aldrich, catalog number: P2287 )
  21. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  22. Wash solution I (see Recipes)
  23. Wash solution II (see Recipes)
  24. Culture Solution (see Recipes)
  25. CFSE (see Recipes)

Equipment

  1. Stereo-microscope with fluorescence capabilities (Olympus, model: SZX16 )
  2. Confocal microscope for detailed analysis (Olympus FV1200 Scanning Laser Confocal Microscope) (if required)
  3. HeraCELL 150 CO2 Incubator set at 37 °C, 5% CO2 (Thermo Fisher Scientific, model: NC0689918 )
  4. Pipettes (P10, P200 and P1000) (Gilson)
  5. Iris scissors (Medicon, catalog number: 202.20.11 )
  6. Forceps (Medicon, catalog number: 07.55.20 )

Procedure

If only external labeling of the larvae is required, follow step A1 from the Internal labeling protocol and then proceed with the external labeling protocol.

  1. Internal labeling of N. brasilienisis iL3 using YO Microspheres
    1. Prepare sufficient Nippostrongylus brasiliensis L3 larvae for experiment by washing larvae three times with 3 ml of PBS in a 15 ml tube and allowing larvae to settle by gravity after each wash. Allowing the larvae to settle by gravity should take approximately 15 min and the larvae should form a pellet at the bottom of the 15 ml tube. This is carried out at room temperature.
      Note: 600 Nippostrongylus brasiliensis L3 larvae are used per injection. By preparing at least 700 Nippostrongylus L3 per injection, it allows for any loss during the labeling procedure.
    2. Transfer the pellet of L3 into a 1.7 ml micro-centrifuge tube using a 1 ml pipette, allow the larvae to form a pellet at the bottom of the tube by gravity for approximately 15 min and then discard any remaining PBS.
    3. Add 500 μl of Wash solution I and incubate at 37 °C in a water-bath for 1 h. Following incubation, discard Wash solution I using a pipette.
    4. Prepare a suspension of 1,500 L3 per ml in Culture Solution by microscopically counting a 10 μl sample of the L3 suspension and adjusting the concentration of larvae with Culture Solution.
    5. Add 1 ml of 1,500 L3 per ml suspension into wells of a 24 well plate. Incubate at 37 °C, 5 % CO2 overnight.
    6. Add 1 μl of YO microspheres to each well and mix by gently swirling the plate.
      Please refer to Note 1 in the Notes section for more information regarding this step.
    7. Incubate for 4 h at room temperature in the dark by placing in a drawer or covering the plate with tinfoil.
      Please refer to Notes 2-3 in the Notes section for more information regarding this step.
    8. Add 1 ml Wash solution II to each well and mix by gently swirling the plate.
    9. Transfer the larvae from each well into 15 ml tubes covered with tin foil to protect from light. Let the larvae settle for approximately 15 min to form a pellet and discard supernatant.
    10. Add 3 ml Wash Solution II to each tube and allow larvae to settle by gravity.
    11. Discard supernatant and repeat at least three times or until the supernatant becomes clear.

  2. External labeling of N. brasilienisis iL3 using CFSE
    1. Transfer the larval pellet in a 1.7 ml micro-centrifuge tube and cover with tin foil to protect from light.
    2. Allow the larvae to settle and discard supernatant using a pipette.
    3. Add 1 μl of 10 mM CFSE to the pellet and mix by gently flicking the micro-centrifuge tube 2-3 times.
    4. Incubate at room temperature for 8 min.
    5. Add 1 ml Wash solution II and mix by inverting 2-3 times.
    6. Allow larvae to settle by gravity and discard supernatant using a pipette.
    7. Repeat wash step at least three times or until the supernatant becomes clear.

  3. In vivo imaging of N. brasilienisis iL3
    1. Prepare a suspension of 600 L3 per 30 μl in PBS by microscopically counting a 10 μl sample of the L3 suspension and adjusting the larval concentration with PBS.
    2. Using an insulin syringe and needle, inject 30 μl of the L3 suspension in the appropriate site of the body intended for study:
      1. Use intra-dermal (i.d.) administration of N. brasilienisis iL3 on the ventral surface (belly skin) or the ear pinnae for imaging of the skin. Hair should be plucked around the site of injection for optimal imaging results (Figure 1).
      2. Use intra-venous (i.v.) administration of N. brasilienisis iL3 for imaging of worms migrating to lung.
        Note: Intra-nasal administration of live larvae does not lead to direct infection and development in the lung. Rather, the infective larvae must first past into the venous blood supply from the airway mucosa and arrive in the lungs 16-18 h later.


        Figure 1. Representative image of intra-dermal administration of N. brasiliensis larvae in the ear pinna with India ink incorporated in the injected solution for visualization purposes only [adapted from Camberis et al. (2013)]

    3. Harvest the tissue of interest
      1. Ear-Euthanise the mouse using an approved form of euthanasia, remove the pinna using scissors and place in a Petri dish containing cold PBS for approximately 15 min to lightly anaesthetize the larvae.
      2.  Belly skin-Euthanise the mouse using an approved form of euthanasia, cut a 1 cm x 1 cm piece of skin around the injection site using scissors, peel skin off and place the skin piece ventral side down in a Petri dish containing cold PBS for approximately 15 min to lightly anaesthetize the larvae. Take care to spread the skin out.
      3. Lung-Euthanise the mouse using an approved form of euthanasia. Expose the thoracic cavity and remove the lungs in one piece and place in a Petri dish containing cold PBS for approximately 15 min to lightly anaesthetize the larvae.
    4. Transfer the tissue onto a glass slide.
      1. Belly skin-Place a cover slip onto the tissue.
      2. Ear-Flatten the ear pinna as much as possible.
      3. Lung-Place lung ventral side up.
    5. Image using fluorescent stereomicroscope or Confocal microscope for more detailed imaging in the tissue.
      For visualizing dyes using the Confocal microscope:
      CFSE 473 nm laser -BP 515/50
      NY 559 nm laser-BP 625/100
      For visualizing dyes using the stereo microscope with fluorescence:
      CFSE BP Excitation filter 495/10-Emission BP 538/45
      NY Excitation filter 545/30-Emission filter BP 610/75

Representative data


Figure 2. Representative Fluorescent microscopy image showing: Top left, Nippostrongylus larvae stained internally with YO microspheres and stained externally with CFSE, Top right, Nippostrongylus larvae stained internally with YO micro beads and Bottom right, CFSE labeled empty larval sheath

Notes

Two thorough reviews of the basic techniques involved in the preparation, handling and use of N. brasiliensis are Kassai (1982) and Camberis et al. (2003).
The protocol outlined above enables reproducible staining of nematodes and their visualization in vivo in tissues for up to 24 h.

  1. N. brasiliensis larvae need to be feeding for the ingestion of fluorescent YO microspheres. In order to activate feeding, larvae need to be incubated at 37 °C in 5% CO2 for a 10-h incubation period. Incubation periods of six hours or less are insufficient to activate the larvae to feed.
  2. The microspheres used in this protocol are fluorescent polystyrene microspheres that have carboxylate groups on their surfaces, which can aid in the covalent coupling of proteins. They can be toxic to the larvae; both the volume of microspheres and incubation times are critical factors in determining whether the larvae will survive. Microsphere volumes greater than 1 μl microspheres are toxic and should be avoided.
  3. The four-hour incubation period of the microspheres with the larvae has been optimized to allow for a signal bright enough for in vivo visualization with minimal toxicity, as larvae can still complete the life cycle through to fecund adults in the gut. In our hands, incubation times greater than 4 h are toxic, however, incubation times less than 4 h do not provide a bright enough signal for in vivo visualization.

Recipes

  1. Wash solution I
    100 ml PBS
    5 ml Penicillin/Streptomicin
    1.5 ml Gentamicin solution
    Can be made in advance and stored for up to one month at 4 °C
  2. Wash solution II
    100 ml PBS
    0.05 ml Tween 20
    Can be made in advance and stored for up to one month at 4 °C
  3. Culture solution
    100 ml DMEM
    10 ml fetal calf serum
    5 ml Penicillin/Streptomicin
    Can be made in advance and stored for up to one month at 4 °C
  4. CFSE
    10 mM CFSE in DMSO
    5.57 mg CFSE in DMSO (1 ml)
    Stored frozen in 10 μl aliquots

Acknowledgments

This protocol was adapted and modified from Hawdon and Schad (1990) and from previous work carried out by Professor Weninger’s Immune Imaging Team at Centenary Institute, Australia. We thank the Hugh Green Cytometry Core for confocal microscopy assistance. This work was supported by The Health Research Council of New Zealand and the Marjorie Barclay Trust.

References

  1. Bouchery, T., Kyle, R., Camberis, M., Shepherd, A., Filbey, K., Smith, A., Harvie, M., Painter, G., Johnston, K., Ferguson, P., Jain, R., Roediger, B., Delahunt, B., Weninger, W., Forbes-Blom, E. and Le Gros, G. (2015). ILC2s and T cells cooperate to ensure maintenance of M2 macrophages for lung immunity against hookworms. Nat Commun 6: 6970.
  2. Camberis, M., Le Gros, G. and Urban, J., Jr. (2003). Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Curr Protoc Immunol Chapter 19: Unit 19 12.
  3. Camberis, M., Prout, M., Tang, S. C., Forbes-Blom, E., Robinson, M., Kyle, R., Belkaid, Y., Paul, W. and Le Gros, G. (2013). Evaluating the in vivo Th2 priming potential among common allergens. J Immunol Methods 394(1-2): 62-72.
  4. Harvie, M., Camberis, M., Tang, S. C., Delahunt, B., Paul, W. and Le Gros, G. (2010). The lung is an important site for priming CD4 T-cell-mediated protective immunity against gastrointestinal helminth parasites. Infect Immun 78(9): 3753-3762.
  5. Hawdon, J. M. and Schad, G. A. (1990). Serum-stimulated feeding in vitro by third-stage infective larvae of the canine hookworm Ancylostoma caninum. J Parasitol 76(3): 394-398.
  6. Kassai, T. (1982). Handbook of Nippostrongylus brasiliensis, Akademia Kiado, Budapest, ISBN-10: 9630529769.

简介

寄生线虫与其宿主之间的相互作用的可视化使得能够更好地理解线虫在其生命周期的感染阶段期间的发展以及宿主对组织中线虫完整性的反应的影响。 这些线虫/宿主相互作用的适当的活体成像迄今为止由于缺乏合适的分子工具或有效的标记试剂而受到阻碍。 在这里,我们提出了线虫寄生虫(Nippostrongylus brasiliensis)( N。brasiliensis )的活标签的技术,其允许在小鼠宿主中可视化寄生虫长达24小时。 外鞘可以用CFSE标记,允许感染性幼虫被鉴定和跟踪,直到呕吐的阶段。 感染性寄生虫的内部标记可以通过摄取NY微球来进行。 蠕虫可以继续被识别长达24小时以下的exshement。 这应适用于其他寄生线虫。

关键字:巴西钩虫, 线虫, 钩虫, 活体成像, 显微镜

材料和试剂

  1. 200μl移液管吸头(Corning,Axygen ,目录号:T-300-L-R-S)
  2. 10μl移液管吸头(Corning,Axygen ,目录号:T-300-R-S)
  3. 1ml移液管吸头(Corning,Axygen ,目录号:T-1000-C-L-R-S)
  4. 1.7ml MaxyClear Snaplock微量离心管(Corning,Axygen ,目录号:MCT-175-C)
  5. 24孔组织培养板(Thermo Fisher Scientific,Falcon ,目录号:353047/08-772-1)
  6. 胰岛素注射器29G,0.3ml超细(BD,目录号:320431)
  7. 15ml锥形离心管(Thermo Fisher Scientific,Falcon ,目录号:352096/14-959-49B)
  8. 显微镜载玻片(Thermo Fisher Scientific,目录号:LBS2951RC)
  9. 培养皿,60mm×15mm(In Vitro Technologies,目录号:FAL351007)
  10. 锡箔(任何杂货店)
  11. N。 brasiliensis L3感染性幼虫(iL3)(Camberis等人,2003)
    注意:Camberis (2003)提供了有关 N的准备的详细评论。 brasiliensis 注意:有几个实验室可以维持生命周期,并且可以通过其出版地址与他们联系。
  12. C57Bl/6J鼠标(或选择的应变)
  13. Fluoresbrite YO羧酸酯微球0.50μm(Polysciences,目录号:18720)
  14. 5(6)-CFDA,SE; CFSE(5-(和-6) - 羧基荧光素二乙酸酯,琥珀酰亚胺酯),混合异构体(Life Technologies,Molecular Probes ,目录号:C1157) 注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:C1157" br />
  15. DMEM,高葡萄糖,丙酮酸盐(Life Technologies,Gibco TM ,目录号:11995-065)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11995-065"
  16. PBS(DPBS,无钙,无镁)(Life Technologies,Gibco TM ,目录号:14190-144) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14190-144"
  17. 青霉素/链霉素10,000U/ml(Life Technologies,Gibco TM ,目录号:15140-122)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"
  18. 庆大霉素溶液10mg/ml(Sigma-Aldrich,目录号:G1272)
  19. 胎牛血清,合格,美国(Life Technologies,Gibco TM ,目录号:26140-079)
    注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:26140-079"
  20. TWEEN 20(Sigma-Aldrich,目录号:P2287)
  21. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  22. 洗涤溶液I(参见配方)
  23. 洗液II(参见配方)
  24. 文化解决方案(参见配方)
  25. CFSE(请参阅配方)

设备

  1. 具有荧光能力的立体显微镜(Olympus,型号:SZX16)
  2. 共聚焦显微镜详细分析(Olympus FV1200扫描激光共聚焦显微镜)(如果需要)
  3. 设置在37℃,5%CO 2(Thermo Fisher Scientific,型号:NC0689918)的HeraCELL 150 CO 2培养箱中。
  4. 移液器(P10,P200和P1000)(Gilson)
  5. 虹膜剪刀(Medicon,目录号:202.20.11)
  6. 钳(Medicon,目录号:07.55.20)

程序

如果只需要外部标记幼虫,按照步骤A1从内部标记协议,然后继续外部标记协议。

  1. 内部标记 N。 brasilienisis iL3使用YO微球
    1. 准备足够的Nippostrongylus brasiliensis L3幼虫进行实验 ?通过在3ml管中用3ml PBS洗涤幼虫三次 允许幼虫在每次洗涤后通过重力沉降。允许 幼虫通过重力沉降需要大约15分钟, 幼虫应在15ml管的底部形成沉淀。这是 在室温下进行 注意:600 Nippostrongylus 每次注射使用巴西丽L3幼虫。准备至少700 ?Nippostrongylus L3每次注射,它允许任何损失期间 标签程序。
    2. 将L3的沉淀转移到1.7ml 微量离心管用1ml移液管,使幼虫形成a 通过重力在管的底部沉淀约15分钟和 ?然后丢弃任何剩余的PBS
    3. 加入500μl的洗涤溶液I 并在37℃在水浴中孵育1小时。孵育后, 丢弃用移液管清洗溶液I.
    4. 准备暂停 通过显微计数10μl在培养物溶液中为1,500L3/ml 样品的L3悬浮液和调整幼虫的浓度 文化解决方案。
    5. 加入1毫升1,500 L3每毫升悬浮液到24孔板的孔中。在37℃,5%CO 2中孵育过夜。
    6. 向每个孔中加入1μlYO微球,并通过轻轻地涡旋板来混合。
      有关此步骤的详情,请参阅备注部分的注释1。
    7. 在室温下在黑暗中通过放入抽屉或用锡箔覆盖板在室温孵育4小时。
      有关此步骤的详情,请参阅备注部分的注意事项2-3。
    8. 向每个孔中加入1ml洗涤溶液II,轻轻旋转平板进行混合。
    9. 将幼虫从每个井转移到15毫升锡覆盖的管 ?箔以保护光。让幼虫沉降约15 以形成沉淀并丢弃上清液。
    10. 向每个管中加入3ml洗涤溶液II,使幼虫通过重力沉降
    11. 弃去上清液并重复至少三次或直到上清液变澄清。

  2. 外部标记 N。 brasilienisis iL3使用CFSE
    1. 转移幼虫颗粒在1.7毫升微离心管,并盖上锡箔,以防止光。
    2. 使幼虫沉降,并使用移液器丢弃上清液
    3. 向沉淀中加入1μl10 mM CFSE,轻轻轻轻微量离心管2-3次,混匀。
    4. 在室温下孵育8分钟。
    5. 加入1ml洗涤溶液II,颠倒混合2-3次
    6. 允许幼虫通过重力沉降,并使用移液管丢弃上清液
    7. 重复洗涤步骤至少三次或直到上清液变澄清。

  3. N的体内成像。 brasilienisis iL3
    1. 通过显微镜在PBS中制备600μL/30μl的悬浮液 计数10μl的L3悬浮液样品并调整幼虫 用PBS浓缩
    2. 使用胰岛素注射器和针, 注射30微升的L3悬浮在身体的适当部位 打算研究:
      1. 使用皮内(i.d.)施用N. 腹部表面(腹部皮肤)或耳廓上的腹膜炎 用于皮肤成像。头发应该在站点周围采摘 注射以获得最佳成像结果(图1)。
      2. 使用静脉内(i.v.)给予N。 brasilienisis iL3用于蠕虫迁移到肺的成像 注意:活的幼虫的鼻内给药不导致直接的 感染和发展。相反,感染性幼虫必须 ?首先进入静脉血供应从气道粘膜和 16-18小时后到达肺部。


        图1.代表性图像 ?皮内施用。 brasiliensis 幼虫在耳廓 其中印度油墨结合在注射溶液中用于可视化 仅为目的 [改编自Camberis (2013)]

    3. 收获感兴趣的组织
      1. 耳 - 安乐死的老鼠使用批准形式的安乐死,删除 ?耳廓使用剪刀和地方在含有冷的PBS的培养皿 大约15分钟,以轻轻麻醉幼虫
      2.  腹部皮肤 - 安乐死的老鼠使用批准形式的安乐死,切 ?使用剪刀在注射部位周围的1cm×1cm的皮肤上, 剥离皮肤,并将皮肤片的腹侧向下放置在培养皿中 ?含有冷PBS约15分钟以轻轻麻醉 幼虫。小心散开皮肤。
      3. 肺安乐死 ?小鼠使用批准形式的安乐死。暴露胸腔 并取出一块中的肺,并放在含有培养皿 冷PBS约15分钟,以轻轻麻醉幼虫。
    4. 将组织转移到载玻片上。
      1. 腹部皮肤 - 将盖玻片放置在组织上。
      2. 耳朵尽可能平整耳廓。
      3. 肺放置肺腹侧向上。
    5. 图像使用荧光立体显微镜或共焦显微镜在组织中更详细的成像。
      使用共焦显微镜可视化染料:
      CFSE 473nm laser -BP 515/50
      NY 559nm laser-BP 625/100
      使用具有荧光的立体显微镜可视化染料:
      CFSE BP激发滤波器495/10-Emission BP 538/45
      NY激发滤光器545/30-发射滤光器BP 610/75

代表数据


图2.代表性荧光显微镜图像,显示:用YO微球体内部染色的顶部左侧,Nippostrongylus幼虫,并用CFSE外部染色,右上部,Nippostrongylus幼虫内部染色YO微球和右下,CFSE标记的空幼虫鞘

笔记

对涉及到的准备,处理和使用的基本技术的两个全面审查。 brasiliensis 是Kassai(1982)和Camberis (2003)。
上述方案使线虫的可重现的染色和它们在体内在组织中的可视化长达24小时。

  1. N。巴西鼠幼虫需要摄食荧光YO微球体。为了激活饲养,需要将幼虫在37℃下在5%CO 2中孵育10小时的孵育期。 6小时或更少的孵化时间不足以激活幼虫进食。
  2. 在该方案中使用的微球是在其表面上具有羧酸根基团的荧光聚苯乙烯微球,其可以帮助蛋白质的共价偶联。它们可能对幼虫有毒;微球的体积和孵育时间是确定幼虫是否将存活的关键因素。微球体积大于1微升的微球是有毒的,应该避免。
  3. 微球与幼虫的四小时孵育期已经被优化以允许足够的信号足够用于体内可视化,具有最小的毒性,因为幼虫仍然可以完成通过成虫的生命周期肠道。在我们的手中,大于4小时的孵育时间是有毒的,然而,小于4小时的孵育时间不提供用于体内可视化的足够明亮的信号。

食谱

  1. 洗液I
    100 ml PBS
    5ml青霉素链霉素 1.5 ml庆大霉素溶液 可以提前做好,并在4°C下保存一个月。
  2. 洗液II
    100 ml PBS
    0.05ml Tween 20 可以提前做好,并在4°C下保存一个月。
  3. 文化解答
    100ml DMEM
    10ml胎牛血清
    5ml青霉素链霉素 可以提前做好,并在4°C下保存一个月。
  4. CFSE
    10mM CFSE的DMSO溶液 5.57mg CFSE在DMSO(1ml)中的溶液 保存在10μl等分试样中冻结

致谢

该协议由Hawdon和Schad(1990)和以前由澳大利亚百年研究所的Weninger免疫成像团队进行的工作进行了修改和修改。我们感谢休格绿色细胞计数核心共焦显微镜协助。这项工作得到了新西兰健康研究委员会和Marjorie Barclay信托的支持。

参考文献

  1. Bouchery,T.,Kyle,R.,Camberis,M.,Shepherd,A.,Filbey,K.,Smith,A.,Harvie,M.,Painter,G.,Johnston,K.,Ferguson, Jain,R.,Roediger,B.,Delahunt,B.,Weninger,W.,Forbes-Blom,E.and Le Gros,G.(2015)。 ILC2和T细胞合作以确保M2巨噬细胞维持针对钩虫的肺免疫。 Nat Commun 6:6970.
  2. Camberis,M.,Le Gros,G。和Urban,J.,Jr。(2003)。 巴西尼日利亚动物模型和Heligmosomoides polygyrus。 < em> Curr Protoc Immunol 第19章:第19单元12.
  3. Camberis,M.,Prout,M.,Tang,S.C.,Forbes-Blom,E.,Robinson,M.,Kyle,R.,Belkaid,Y.,Paul,W.and Le Gros,G。 评估常见过敏原中体内 Th2引发潜力。 J Immunol Methods 394(1-2):62-72。
  4. Harvie,M.,Camberis,M.,Tang,S.C.,Delahunt,B.,Paul,W。和Le Gros,G。(2010)。 肺是预防胃肠蠕虫寄生虫的CD4 T细胞介导的保护性免疫的重要位点。 Infect Immun 78(9):3753-3762。
  5. Hawdon,J.M。和Schad,G.A。(1990)。 体外血清刺激喂养 由第三阶段感染性幼虫犬钩虫Ancylostoma caninum。 Parasitol 76(3):394-398。
  6. Kassai,T。(1982)。 "Nippostrongylus brasiliensis 手册,Akademia Kiado,Budapest,ISBN-10:9630529769.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bouchery, T., Camberis, M. and Gros, G. L. (2016). Dye Labeling of Live Nippostrongylus brasiliensis Larvae for Visualization in Host Tissue. Bio-protocol 6(4): e1737. DOI: 10.21769/BioProtoc.1737.
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