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