Magnetic-Activated Cell Sorting: A Method to Isolate c-Kit Positive Cells
筆記録
– 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, harvest the leg bones from a euthanized mouse. Clean the tissues from the bones with a scalpel. Then, place the bones in cold PBS on ice and crush the bones with a pestle. After this, filter the cell suspension through a 70-micrometers cell strainer into a 50-milliliter screw cap tube with a 10-milliliter pipette. Wash the mortar and pestle multiple times with cold PBS, and add the cell suspension to the 50-milliliter tube.
Next, centrifuge the bone marrow and remove the supernatant with vacuum and glass pipette. Resuspend the cell pellet in five milliliters of PBS. Using a pipette, slowly add the suspended bone marrow to the top of room temperature polysucrose.
After this, spin the polysucrose at 400 G's for 20 minutes with the brake turned off. After cetrifugation, collect the cloudy total mononuclear cell interface with a pipette and transfer it to a new 15-milliliter tube. Bring the volume to 10 milliliters with PBS and remove 20 microliters for counting.
After cetrifugation, discard the supernatant and place the pellet on ice. Next, resuspend the cell pellet in PBS with EDTA and BSA. Add CD117 microbeads to the cell suspension and vortex to mix. After this, incubate the suspension for 10 minutes at 4 degrees Celsius. Then, bring the volume up to 10 milliliters with more PBS with EDTA and BSA and centrifuge at 1,200 G's at 4 degrees Celsius for five minutes.
Use the vacuum to remove the supernatant and suspend the cell pellet in PBS with EDTA and BSA. Then, add three milliliters of PBS with EDTA and BSA to a magnet stand with a magnetic column and allow it to flow into a collection tube. After the buffer finishes flowing through the column, add the cell suspension and allow it to flow through the column into the collection tube.
Next, add 5 more milliliters of PBS with EDTA and BSA to the column and flush it immediately with the plunger. Remove the plunger and repeat the flush before counting the cells. After centrifuging the cells, remove the supernatant and suspend the cell pellet in complete IMDM for culture. Culture the cells overnight at 37 degrees Celsius with 5% carbon dioxide.
