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NOTE: This protocol describes the isolation of HT-29 cells (human colon cancer cell line) from PBS or from artificial mixtures of HT-29 cells and peripheral blood. The same experiment was performed with two additional cell lines (LNCaP and VCaP) and can theoretically be performed with all cells expressing EpCAM.
1. Preparation of Target Cells
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	<li>Cell culture and labeling of cells 
	NOTE: In this ...

functionalized wire-based target cell isolation: an ex vivo technique to isolate cancer cells from spiked peripheral blood

Source: Chen S. et al. <a href="https://www.jove.com/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of EpCAM-expressing epithelial cancer cells from peripheral blood samples. The technique involves the use of a medical-grade wire functionalized with EpCAM&#160;antibodies to capture the target cells. The captured cells are then subsequently extracted into a buffer solution.

<img alt="Figure 1" src="/files/ftp_upload/20395/20395_Figure1.jpg" /> 
Figure 1: Visualizing labeled cells.&#160;(A,E)&#160;Cells before charging: Cells are labeled with CFSE and counterstained with a DNA intercalating dye showing a range of CFSE (green) signal intensity.&#160;(B,F)&#160;Labeled cells captured on the wire.&#160;(C,G)&#160;Wire after cell-detachment ...

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)
In this ...

Epithelial cell adhesion molecules, or EpCAMs, are surface transmembrane glycoproteins overexpressed in rapidly proliferating epithelial cells in prostate cancers. To isolate EpCAM-expressing cells, begin with a medical-grade wire bearing a functionalized gold-coated tip. Functionalization layers a polymeric gel containing covalently bound EpCAM-specific antibodies.
Immerse the wire into a peripheral blood suspension containing fluorescently labeled cancer cells. Ensure that the functionalized part remains dipped in the blood suspension. The EpCAMs on the cancer cells recognize and bind to their specific antibodies functionalized on the wire.
Wash with a buffer to remove any unbound cells from the wire surface. Bend the wire and mount it on a buffer-containing glass slide, with the functionalized part immersed in the buffer. Use a fluorescence microscope to check bright fluorescence from cell cytoplasm and nuclei and confirm the presence of captured cells on the wire.
Treat the wire with a release buffer. The proteolytic enzymes in the buffer cleave the polymeric gel, releasing the antibody-cancer cell complexes into the buffer. Centrifuge the wire assembly to collect the released antibody-cell complexes in the pellet. Remove the wire and the enzyme-containing supernatant. Resuspend the target cell-containing pellet in a suitable medium.

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

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