三维球状体共培养侵袭研究

Eldo T Verghese; Thomas A Hughes

doi:10.21769/BioProtoc.1373

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Peer-reviewed

Three Dimensional Spheroid Co-culture Invasion Assay

三维球状体共培养侵袭研究

EV Eldo T Verghese

TH Thomas A Hughes email

发布: 2015年01月05日第5卷第1期 DOI: 10.21769/BioProtoc.1373 浏览次数: 13130

评审: Lin FangAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Pathology, featuring study using the protocol.

Nov 2013

实验方案合集

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Abstract

The assay was developed to investigate the impact of stromal cells of different types (in our case breast cancer associated fibroblasts stably manipulated to modify expression of genes of interest) on the invasive capacity of epithelial cancer cells (in our case breast cancer cell lines) (Verghese et al., 2013). Typical two dimensional invasion assays do necessarily account for the presence of extracellular matrix that is present around the stromal and tumour cells in vivo and therefore cellular behaviour within these cultures may be non-physiological. This spheroid assay was developed to attempt to replicate more closely the environment that is present around breast cancer stromal and tumour cells in actual tumours (Verghese et al., 2013). Extra cellular matrix composed of both collagen IV and collagen I is included and fibroblasts and epithelial cells are given the opportunity to develop “physiological” interactions (Verghese et al., 2013; Hooper et al., 2006). The method was developed from Nowicki et al. (2008), and we have published data using it in Verghese et al. (2013).

Materials and Reagents

Epithelial cells and fibroblasts [see Verghese et al. (2013) for the type we have used, although many others may be applicable]
Growth factor reduced matrigel (BD, catalogue number: 356231 )
Collagen-1 (BD, catalog number: 354236 )
Fetal calf serum (FCS) (Sigma-Aldrich, catalog number: F7524 )
DMEM (Life Technologies, Invitrogen^TM, catalog number: 31966 )
MEM powder (Sigma-Aldrich, catalog number: M0268 )
1 M N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (pH 7.5)
NaHCO₃
Rabbit anti-cytokeratin (Abcam, catalog number: Ab9377 )
Zymed antibody diluent (Life Technologies, catalog number: 003218 )
DPX (Sigma-Aldrich, catalog number: 317616 ) (a mounting agent)
Mayer’s haematoxylin (any supplier)
Scott’s tap water substitute (any supplier)
Eosin (any supplier)
Formalin (any supplier)
Ethanol (any supplier)
Xylene (any supplier)
Collagen-1/Matrigel mix (see Recipes)
Collagen medium (see Recipes)

Equipment

Tissue culture incubator (37 °C, humidified air/5% CO₂) (Panasonic Corporation, Sanyo)
35 mm petri dish (ibidi GmbH, catalog number: 81158 )
E1000 Eclipse microscope (Nikon Corporation)
Superfrost Plus slides and cover slips (Menzel Glaser)
Access retrieval unit (The Menarini Group) (a machine performing automated antigen retrieval giving enhanced reproducibility)
Automated stainer (IntelliPATH) (a machine performing automated immunohistochemistry protocols giving enhanced reproducibility)

Software

Photoshop (ADOBE)

Procedure

English

中文翻译

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如何引用

Verghese, E. T. and Hughes, T. A. (2015). Three Dimensional Spheroid Co-culture Invasion Assay. Bio-protocol 5(1): e1373. DOI: 10.21769/BioProtoc.1373.