A single-cell suspension from the lymphoid tissue of interest is labeled with BD IMag™ anti-mouse CD117 Particles - DM according to the Magnetic Labeling Protocol. This labeled cell suspension is then placed within the magnetic field of the BD IMag™ Cell Separation Magnet (Cat. no. 552311). Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off (negative fraction). The tube is then removed from the magnetic field for resuspension of the positive fraction. The separation is repeated twice to increase the purity of the positive fraction. The magnetic separation steps are diagrammed in the Separation Flow Chart. After the positive fraction is washed, the small size of the magnetic particles allows the positive fraction to be further evaluated in downstream applications such as flow cytometry.
MAGNETIC LABELING PROTOCOL
1. Prepare a single-cell suspension from the lymphoid tissue of interest according to standard laboratory procedures. Remove clumps of cells and/or debris by passing the suspension through a 70-mm nylon cell strainer.
2. Dilute BD IMag™ Buffer (10X) (Cat. no. 552362) 1:10 with sterile distilled water or prepare 1X BD IMag™ buffer by supplementing Phosphate Buffered Saline with 0.5% BSA, 2 mM EDTA, and 0.09% sodium azide. Store at 4˚C.
3. Wash cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.
4. Vortex the BD IMag™ anti-mouse CD117 Magnetic Particles - DM thoroughly, and add 50 μl of particles for every 10 million total cells.
5. MIX THOROUGHLY. Refrigerate at 6°C to 12°C for 30 minutes.
6. Bring the BD IMag™-particle labeling volume up to 10 to 80 million cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the Cell Separation Magnet. Incubate at room temperature for 8-10 minutes.
7. With the tube on the Cell Separation Magnet, carefully pipette off the supernatant. This supernatant contains the negative fraction.
8. Remove the tube from the Cell Separation Magnet, and add 1 ml of 1X BD IMag™ buffer. Gently resuspend cells by pipetting up and down, and return the tube to the Cell Separation Magnet for another 2 - 4 minutes.
9. With the tube on the Cell Separation Magnet, carefully pipette off the supernatant and discard.
10. Repeat Steps 8 and 9.
11. After the final wash step, resuspend the positive fraction in an appropriate buffer or media, and proceed with desired downstream application(s).
NOTE: The concentration of BD IMag™ anti-mouse CD117 Particles - DM suggested in the protocol has been optimized for the purification of CD117-positive cells from mouse bone marrow. When labeling target cell populations present at lower frequencies, fewer BD IMag™ particles can be used. Conversely, when labeling target cell populations that are present at higher frequencies, more particles should be used. To determine the optimal concentration of the BD IMag™ anti-mouse CD117 Particles - DM for a particular application, a titration in two-fold increments is recommended.
NOTE: Avoid nonspecific labeling by working quickly and keeping incubation times as recommended.