Melanoma Stem Cell Isolation: A Method to Obtain CD133 Positive and Negative Melanoma Cells Using Magnetic-activated Cell Sorting

- Melanoma harbors a small subpopulation of cancer stem cells possessing both self-renewal and tumor initiation capabilities. CD133 or Prominin-1, a pentaspan transmembrane glycoprotein, is one of the key markers expressed by metastatic melanoma cancer stem cells. To isolate CD133-positive cells, begin with a suspension of primary melanoma cells in an appropriate buffer.

Incubate with a blocking reagent containing proteins that block non-specific binding sites on the cell surface. Treat the cells with biotinylated CD133 primary antibodies. These antibodies bind to CD133 antigens expressed on CD133-positive cells. Now, add anti-biotin magnetic microbeads to the suspension. Incubate for the microbeads to bind to the biotin-labeled cells.

Finally, pass the incubated suspension through a column placed in a magnetic field. With magnetic influence, all CD133-positive cells labeled with the microbeads stick to the column, while the unlabeled cells pass through. Collect these unlabeled CD133-negative cells. Remove the column from the magnetic field. Flush the contents using a suitable buffer to elute and collect CD133-positive cells.

In the following protocol, we will show the isolation of