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
发布: 2016年02月20日第6卷第4期 DOI: 10.21769/BioProtoc.1737 浏览次数: 8715
评审: Ivan ZanoniHongwei HanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
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
- 200 μl pipette tips (Corning, Axygen®, catalog number: T-300-L-R-S )
- 10 μl pipette tips (Corning, Axygen®, catalog number: T-300-R-S )
- 1 ml pipette tips (Corning, Axygen®, catalog number: T-1000-C-L-R-S )
- 1.7 ml MaxyClear Snaplock Microcentrifuge Tube (Corning, Axygen®, catalog number: MCT-175-C )
- 24-well Tissue culture plate (Thermo Fisher Scientific, FalconTM, catalog number: 353047/08-772-1 )
- Insulin syringe 29 G, 0.3 ml ultrafine (BD, catalog number: 320431 )
- 15 ml Conical Centrifuge Tubes (Thermo Fisher Scientific, FalconTM, catalog number: 352096 / 14-959-49B )
- Microscope slides (Thermo Fisher Scientific, catalog number: LBS2951RC )
- Petri dish, 60 mm x 15 mm (In Vitro Technologies, catalog number: FAL351007 )
- Tin-foil (any grocery store)
- 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. - C57Bl/6J Mouse (or strain of choice)
- Fluoresbrite® YO Carboxylate Microspheres 0.50 µm (Polysciences, catalog number: 18720 )
- 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”. - DMEM, high glucose, pyruvate (Life Technologies, GibcoTM, catalog number: 11995-065 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065”. - 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”. - 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”. - Gentamicin solution 10 mg/ml (Sigma-Aldrich, catalog number: G1272 )
- 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”. - TWEEN® 20 (Sigma-Aldrich, catalog number: P2287 )
- Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
- Wash solution I (see Recipes)
- Wash solution II (see Recipes)
- Culture Solution (see Recipes)
- CFSE (see Recipes)
Equipment
- Stereo-microscope with fluorescence capabilities (Olympus, model: SZX16 )
- Confocal microscope for detailed analysis (Olympus FV1200 Scanning Laser Confocal Microscope) (if required)
- HeraCELL 150 CO2 Incubator set at 37 °C, 5% CO2 (Thermo Fisher Scientific, model: NC0689918 )
- Pipettes (P10, P200 and P1000) (Gilson)
- Iris scissors (Medicon, catalog number: 202.20.11 )
- Forceps (Medicon, catalog number: 07.55.20 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
免疫学 > 动物模型 > 小鼠
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