Leukocytes are labeled with BD IMag™ anti-mouse CD8a Particles - DM according to the Magnetic Labeling Protocol. This labeled cell suspension is then placed within the magnetic field of 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-µm 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. Place on ice.
Although our experience indicates that the use of Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™)(Cat. No. 553141/553142) is not required for optimal cell separation, some laboratories may want to use it in their studies. If adding Mouse BD Fc Block, proceed to Step 3. If not adding Mouse BD Fc Block, proceed to Step 4.
3. Add Mouse BD Fc Block at 0.25 µg/10^6 cells, and incubate on ice for 15 minutes.
4. Wash cells with at least an equal volume of 1X BD IMag buffer, and carefully aspirate all the supernatant.
5. Vortex the BD™ IMag anti-mouse CD8a Particles - DM thoroughly, and add 50 µl of particles for every 10^7 total cells.
6. MIX THOROUGHLY. Refrigerate at 6°C - 12°C for 30 minutes.
7. Bring the BD IMag-particle labeling volume up to 1 - 8 × 10^7 cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the BD IMag™ Cell Separation Magnet. Incubate at room temperature for 6 - 8 minutes.
8. With the tube on the BD IMag™ Cell Separation Magnet, carefully aspirate off the supernatant. This supernatant contains the negative fraction.
9. Remove the tube from the BD IMag™ Cell Separation Magnet, and add 1X BD IMag buffer to the same volume as in Step 7. Gently resuspend cells by pipetting briefly, and return the tube to the BD IMag™ Cell Separation Magnet for another 2 - 4 minutes.
10. With the tube on the BD IMag™ Cell Separation Magnet, carefully aspirate off the supernatant and discard.
11. Repeat Steps 9 and 10.
12. After the final wash step, resuspend the positive fraction in an appropriate buffer and at an appropriate concentration for further analysis.
NOTE: Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.