Metastatic Cancer Cell Colony Isolation from Chicken CAM: A Procedure to Isolate Cancer Cells from Shell-less Egg Cultures

Protokol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolation of Metastatic Colonies

1. At day 5 post injection, remove embryos from the incubator and inspect the embryo CAMs for metastatic colony distribution. Identify the embryos with uniform colony distribution in which compact (or overly invasive) colonies are present.
   NOTE: The process of culturing chicken embryos is not sterile, therefore, all the screening steps must be performed under highly clean conditions to avoid future tissue culture ....

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Sonuçlar

Figure 1. Representative results for different steps of the screening protocol. (A) Metastatic colonies formed by heterogenous (library transduced cancer cell line HEp3 cells), 5 days post injection. Red arrow shows compact colony (potential positive hit) that should be excised. (B) Metastatic colonies formed by one of the isolated screen hits (<.......

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Malzemeler

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tubes.	Corning	CLS430791-500EA
18 gauge x 1 1/2 BD precision needle	BD	BD305196	we use 1.2mm x 40mm, it is possible to use shorter needles if preferred
Benchtop centrifuge.	many sources are available		Any TC compatible centrifuge that can be used to spin down the cells is suitable
Collagenase	Sigma	C0130-100MG
Cotton swabs	many sources are available		must be sterilized before use
Culture media appropriate for the cell lines used	many sources are available		We grow HT1080, HEp3 and b16 cell lines in DMEM, 10% FBS media
Egg incubator	many sources are available		An exact model that is necessary depends on the scale of the screen. Available sources are MGF Company Inc., Savannah, GA, or Lyon Electric Company Inc., Chula Vista, CA
Eppendorf tubes , 1.5ml	Sigma	T4816-250EA
Fertilized White Leghorn eggs	any local supplyer
Fine forceps	many sources are available		must be sterilized before use
Image analysis software			We use Nikon Elements
PBS (1x)	many sources are available
Plastic weighting dishes	Simport	CA11006-614	dimensions are 78x78x25mm; many other sources are available
Small surgical scissors	many sources are available		must be sterilized before use
Stereo fluorescent microscope			We use Zeiss Lumar v12