Magnetic-Activated Cell Sorting: A Method to Isolate c-Kit Positive Cells

- c-Kit, or CD117, is a tyrosine kinase transmembrane receptor expressed by hematopoietic stem or progenitor cells present in the bone marrow and blood. It is also a marker for detecting acute myeloid leukemia.

To isolate c-Kit positive cells, harvest murine leg bones and crush them to release the marrow from within. Filter the marrow suspension to remove any bone fragments. Layer the filtrate containing bone marrow cells on a density gradient medium, and centrifuge to separate mononuclear cells from other fractions.

Collect the cloudy layer of mononuclear cells in a tube containing microbeads conjugated to anti-CD117 mouse antibodies. Incubate for the antibody-bead complexes to bind to the CD117 receptors on c-Kit positive cells. Pass the incubated suspension through a magnetic column placed in a magnetic field. All c-Kit positive cells labeled with the magnetic microbeads remain in the column, while the unlabeled cells pass through.

Remove the column from the magnetic field, and flush the contents using a suitable buffer to elute c-Kit positive cells. In the following protocol, we will show c-Kit positive cells' isolation from the bone marrow of a transgenic mouse.

- To begin, ha