In summary, the PE rat anti-mouse CD49b antibody stains NK and NK-T cells. After washing away excess antibody, BD IMag™ Anti-PE Particles 2 - DM are added to the cell suspension and bind the cells bearing the PE-conjugated antibody. The tube containing this labeled cell suspension is then placed within the magnetic field of the BD IMag™ Cell Separation Magnet. Positive selection or depletion is then performed. Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off (depleted or negative fraction). The tube is then removed from the magnetic field for resuspension of the positive fraction. The selections are repeated twice to increase the purity of the positive fraction. For clarification of the procedure, the magnetic separation steps are diagrammed in the Positive Selection Flow Chart. The small size of the BD IMag™ particles allows the positive fraction to be further evaluated in downstream applications such as flow cytometry.
MAGNETIC LABELING AND SEPARATION PROTOCOL
1. 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.
2. Aseptically prepare a single-cell suspension from the peripheral lymphoid tissue of interest. Remove clumps of cells and/or debris by passing the suspended cells through a 70-µm nylon cell strainer. NK cells are fragile and therefore should be prepared in tissue culture medium [e.g., Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum and L-glutamine].
3. Count the cells. If the concentration is between 1 x 10e7 and 2 x 10e7 cells/ml, then proceed to Step 4. If cells are more dilute than 1 x 10e7 cells/ml, then spin down the cells and resuspend them in tissue culture medium at a concentration of 2 x 10e7 cells/ml.
4. Optional: Add BD Mouse Fc Block™ purified anti-mouse CD16/CD32 (Cat. No. 553141) at 0.25 µg per 1 x 10e6 cells, and incubate on ice for 15 minutes.*
5. Add the BD IMag™ PE Rat Anti-Mouse CD49b at 5 µl per 1 x 10e6 cells, and refrigerate for 15 minutes at 6-12°C.†
6. Wash the labeled cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate ALL the supernatant.
7. Vortex the BD IMag™ Anti-PE Particles 2 - DM thoroughly, and add 5 µl of particles for every 1 x 10e6 total cells.
8. Mix thoroughly. Refrigerate for 30 minutes at 6-12°C.†
9. Bring the labeling volume up to 2 to 8 x 10e7 cells/ml with 1X BD IMag™ buffer.
10. Immediately place the tube onto the Cell Separation Magnet and incubate for 6 to 8 minutes.
11. With the tube on the Cell Separation Magnet, carefully aspirate the supernatant. This supernatant is considered the Negative (or NK cell-depleted) Fraction.
12. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 9. Gently resuspend the cells by pipetting up and down, and return the tube to the Cell Separation Magnet for another 2 to 4 minutes. Longer inclubation time will increase the percentage of NK-T cells in the positive fraction.
13. With the tube on the Cell Separation Magnet, carefully remove the supernatant (wash fraction) and discard.
14. Repeat Steps 12 and 13.
15. After the final wash step, remove the tube from the Cell Separation Magnet. Resuspend the Positive Fraction in an appropriate buffer or culture medium, and proceed with desired downstream application(s), including flow cytometry.
NOTES:
* The use of BD Mouse Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 in step 4 can increase the purity and recovery of the NK cells by up to 5%. Please note that this results in enriched NK cells that may have purified anti-mouse CD16/CD32 mAb bound to their surface, which may affect the function of those NK cells.
† Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.
