Invadopodia Detection and Gelatin Degradation Assay
侵足检测和明胶降解分析
发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.997 浏览次数: 22907
评审: Lin FangFanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
Abstract
This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia and their activity is simultaneously assessed and quantified by a fluorescent microscopy-based assay.
Keywords: Cancer (癌症)
Invasion (入侵)
Invadopodia (侵袭性伪足)
Metastasis (转移)
Gelatin (明胶)
Materials and Reagents
- Cells (i.e. SCC61 head and neck carcinoma cells)
- Tissue culture media (i.e. DMEM) (Mediatech, catalog number: 10013CV )
- Sterile PBS
- Trypsin 0.05%/EDTA (Life Technologies, catalog number: 25300054 )
- Penicillin/Streptomycin solution (Omega Scientific, catalog number PS-20 )
- Ethanol (Decon Labs, catalog number: 2801 )
- Forceps Dumont #5 (Fine Science Tools, catalog number: 11252-20 )
- 25% Glutaraldehyde (Polysciences, catalog number: 00376-500 )
- Sucrose (Fisher Scientific, catalog number: S3500 )
- Sodium Borohydride (Fisher Scientific, catalog number: S67825 )
- 16% Formaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
- Gelatin from pig skin, Oregon green 488 conjugate (Life Technologies, catalog number: G13186 )
- Alexa Fluor 568 Phalloidin (Life Technologies, catalog number: A12380 )
- Mounting medium, Vectashield with DAPI (Vector Laboratories, catalog number: H-1200 )
- Bovine Serum Albumin fraction V (EMD Millipore, catalog number: 2980-1KG )
- Triton X-100 (Promega corporation, catalog number: H5141 )
- Parafilm (Bemis Company, catalog number: PM996 )
Equipment
- Round 18 mm diameter glass coverslips (0.13-0.17 mm thickness) (Carolina Biological Supply Company, catalog number: 633033 )
- Microscope glass slides (75 x 25 mm) (Thermo Fisher Scientific, catalog number: 1255015 )
- 12-well Microplates (Corning, catalog number: 3513 )
- Thermomixer or water bath set at 60 °C
- Tissue culture incubator
- Hemocytometer (LabSource, catalog number: 0267154 ) http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/counting-cells-in-a-hemacytometer.html
- Fluorescence Microscope
- Vacuum source
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Díaz, B. (2013). Invadopodia Detection and Gelatin Degradation Assay. Bio-protocol 3(24): e997. DOI: 10.21769/BioProtoc.997.
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Diaz, B., Yuen, A., Iizuka, S., Higashiyama, S. and Courtneidge, S. A. (2013). Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia. J Cell Biol 201(2): 279-292.
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分类
癌症生物学 > 通用技术 > 细胞生物学试验
癌症生物学 > 侵袭和转移 > 细胞生物学试验
细胞生物学 > 细胞成像 > 荧光
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