JoVE Encyclopedia of Experiments
Cancer Research
0 views • 4:31 min • April 30th, 2023
- Chemotherapeutic treatment for tumors generally targets highly proliferative cells, leaving behind a small subset of the stem cell population, leading to the tumor's recapitulation. These stem cells are called the side population. Since the side population lacks specific cell markers, we use a dye efflux assay to isolate them from other tumor cells.
To begin, add Hoechst dye to the isolated zebrafish tumor cells. Hoechst enters the nucleus and binds to the A-T rich region in the minor groove of DNA. The side population of cells has a higher concentration of ABC transporters on the surface, leading to dye's rapid efflux outside the cell, displaying low fluorescence.
Take another vial of Hoesct-treated tumor cells and add verapamil to it. This vial acts as a control, because verapamil blocks the ABC transporters causing a reduction in hoechst efflux, therefore, increasing fluorescence in the side population.
Incubate both the control and sample cells in the dark. Next, counter stain cells with propidium iodide that enter the nucleus of dead cells exclusively and intercalate between DNA bases, staining the dead cells red. Finally, sort the cells using a fluorescence activated cell s
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