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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

Isolation of c-Kit⁺ Cells from Bone Marrow

1. Sacrifice the mice according to institutional guidelines.
   NOTE: Here, the mice were exposed to carbon dioxide (CO₂) at an according to the AVMA recommended flow rate until death was determined by the cessation of respiration and heartbeat, followed by cervical dislocation.
2. Harvest the leg bones from the mouse — two femurs, two tibias, two iliacs. Clean off all tissues from the bones by scraping away with a scalpel. Put the leg bones in cold 1x PBS on ice — 5 mL in a 15 mL tube. Pour the contents of the tube into a mortar and crush it with a pestle.
3. Filter the cell suspension through a 70 µm cell strainer into a 50 mL screw cap tube with a 10 mL pipette attached, using a pipette. Wash the mortar and pestle multiple times with cold 1x PBS, collecting and adding cell suspension to the 50 mL tube. Spin down bone marrow at 1,200 x g at 4 °C for 5 min. Remove the supernatant via a vacuum with a glass pipette attached. Suspend the cell pellet in 5 mL of 1x PBS with a pipette.
4. With a pipette, slowly add suspended bone marrow to the top of 5 mL of room temperature (the temperature is critical) polysucrose, taking great care not to disturb the interface. Spin at 400 x g at room temperature for 20 min with the brake turned off.
5. Collect the cloudy total mono-nuclear cell (TMNC) interface with a pipette and put it into a new 15 mL tube. Bring the volume up to 10 mL with 1x PBS for the wash, taking 20 µL for counting, and spin at 1,200 x g at 4 °C for 5 min. Discard the supernatant and save the pellet on ice for step 4.6.1.
6. Perform c-Kit⁺ cell isolation.
   1. Follow a microbead kit protocol for the c-Kit⁺ cell isolation. Resuspend the cell pellet in 80 µL of 1x PBS + EDTA + BSA to 10⁷ cells. To the cell suspension, add 20 µL of CD117 MicroBeads/10⁷ total cells. Mix well by vortexing and incubate for 10 min at 4 °C. Bring the volume up to 10 mL by adding 1x PBS + EDTA + BSA and spin at 1,200 x g at 4 °C for 5 min.
   2. Remove the supernatant via a vacuum and suspend the cell pellet in 500 µL/10⁸ total cells in 1x PBS + EDTA + BSA. Using the magnet stand with the magnetic column attached, add 3 mL of 1x PBS + EDTA + BSA and allow it to flow through via gravity into a 15 mL collection tube.
   3. Once the first buffer addition has finished passing through the column, add the cell suspension to the column, allowing it to flow through via gravity into the 15 mL collection tube. This flowthrough is the undesired cell fraction.
   4. Wash the column 3 times with 3 mL 1x PBS + EDTA + BSA each time, allowing it to flow through via gravity into the collection tube. Remove the column from the magnet and place onto the top of a 15 mL tube.
   5. Add 5 mL of 1x PBS + EDTA + BSA and immediately flush it with the plunger provided with the column. Remove the plunger and repeat the flush with an additional 5 mL of 1x PBS + EDTA + BSA. Count the cells in the total volume using standard methods.
   6. Spin down the cells at 1,200 x g at 4 °C for 5 min. Remove the supernatant and resuspend the pellet in complete IMDM (IMDM + 10% FBS + IL-6 [10 ng/mL] + mSCF [50 ng/mL] + FLT3 ligand [20 ng/mL] + IL-3 [10 ng/mL]) for culture. Culture these cells overnight in 2 x 10⁶ cells/1 mL of cell IMDM complete media by placing them in an incubator at 37 °C and 5% CO₂.