三维侵袭实验

Wen-Hao  Yang; Muh-Hwa Yang

doi:10.21769/BioProtoc.885

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Peer-reviewed

Three-dimensional Invasion Assay

三维侵袭实验

Wen-Hao Yang

Muh-Hwa Yang email

发布: 2013年09月05日第3卷第17期 DOI: 10.21769/BioProtoc.885 浏览次数: 12960

评审: Lin FangFanglian HeAnonymous reviewer(s)

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Cover of Nature Cell Biology, featuring study using the protocol.

Apr 2012

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Abstract

The invasive ability of cancer cells is a crucial function for cancer metastasis and the surrounding microenvironment of cancer cells in living tissues is three-dimension (3D). Therefore, to establish an in vitro invasion assay in a 3D system to predict cancer invasive ability is valuable in the research for cancer metastasis. Here, we describe a 3D invasion assay for observing the morphology and comparing the invasive ability of cancer cells in artificial 3D environments (Yang et al., 2012). Collagen I gels are used to cover on the top of cancer cells attached on coverslip glass dish and medium containing FBS is added as a chemoattractant. After incubation for a suitable time, the cells are fixed and stained. The invasion index can be calculated and the morphology can be imaged with a laser confocal microscope.

Keywords: 3D invasion (3d入侵)

Collagen gel (胶原凝胶)

Invasion index (侵袭指数)

Materials and Reagents

Cell lines: OECM-1(Huang et al., 2004) and FaDu (ATCC^® HTB-43 ^TM)
0.1% Trypsin-EDTA (Life Technologies, Gibco^®, catalog number: 15400 )
0.1 mg/ml poly-L lysine (Sigma-Aldrich, catalog number: P9404-25MG )
PBS
FBS (Thermo Fisher Scientific, catalog number: SH30071.03 )
PureCor^® bovine collagen solution (Advance Biomatrix Inc., catalog number: 5005-B )
1 M NaOH solution
5x RPMI medium
3% Paraformaldehyde (Sigma-Aldrich, catalog number: P6148-500G )
0.5% Triton X100 (Bionovas, catalog number: 56-81-5 )
Alexa Fluor^® 488 Phalloidin (Life Technologies, catalog number: A12379 )
DAPI (Sigma-Aldrich, catalog number: D8417 )
1% BSA in PBS
1.8 mg/ml collagen I mix solution (see Recipes)