The detailed Magnetic Labeling and Enrichment Protocol follows. In summary, the Human Memory CD8 T Cell Enrichment Cocktail simultaneously stains naïve T cells, CD4 T cells, NK cells, γ/δ T cells, monocytes, DCs, granulocytes, platelets, and erythroid cells. After washing away excess antibody, BD IMag™ Streptavidin Particles Plus - DM are added to the cell suspension and bind the cells bearing the biotinylated antibodies. The tube containing this labeled cell suspension is then placed within the magnetic field of the BD™ IMagnet (Cat. No. 552311). Negative selection is then performed to enrich for the unlabeled memory CD8 T Cells. Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off and retained (enriched fraction). The negative selection is repeated twice to increase the yield of the enriched fraction. If greater purity is required, negative selection may be performed on the enriched fraction. For clarification of the procedure, the magnetic separation steps are diagrammed in the Enrichment Flow Chart. The positive and enriched fractions can be evaluated in downstream applications such as flow cytometry and tissue culture. The biotinylated antibodies in the Human Memory CD8 T Cell Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for the enrichment of memory CD8 T cells from PBMC.
MAGNETIC LABELING AND ENRICHMENT PROTOCOL
1. Prepare 1X BD IMag™ buffer: Dilute BD IMag™ Buffer (10X) (Cat. No. 552362) 1:10 with sterile distilled water or prepare Phosphate
Buffered Saline (PBS) supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide.
2. Prepare PBMC from anti-coagulated human blood, preferably by density gradient centrifugation using Ficoll-Paque™.*
3. Count the cells, and resuspend them in 1X BD IMag™ buffer at a concentration of 50 x 10^6 cells/ml.
4. Add the Human Memory CD8 T Cell Enrichment Cocktail at 5 µl per 1 x 10^6 cells, and incubate at room temperature for 15 minutes. Avoid
nonspecific labeling by working quickly and adhering to recommended incubation times.
5. Wash the labeled cells with a 10X excess volume of 1X BD IMag™ buffer, centrifuge at 300 × g for 10 minutes, and carefully aspirate ALL
the supernatant.
6. Vortex the BD IMag™ Streptavidin Particles Plus - DM thoroughly, and resuspend the cell pellet in 7.5 µl of particles per 1 x 10^6 cells.
Please note that this volume of IMag Streptavidin Particles is higher than that used in most BD IMag™ enrichment sets and that this
Set contains a greater total volume of these particles to account for this difference.
7. MIX THOROUGHLY. Incubate at room temperature for 30 minutes. Avoid nonspecific labeling by working quickly and adhering to
recommended incubation times.
8. Bring the labeling volume up to 20 to 80 x 10e6 cells/ml with 1X BD IMag™ buffer.
9. Transfer the labeled cells to a 12 x 75 mm round-bottom test tube, maximum volume added not to
exceed 1.0 ml. Place this positive-fraction tube on the BD IMagnet™ (horizontal position) for 6 minutes.
• For greater volume, divide the cells into multiple 12 x 75 mm round-bottom test tubes or transfer the cells to a 17 x 100 mm
round-bottom test tube, maximum volume added not to exceed 3.0 ml. Place this positive-
fraction tube on the BD IMagnet™ (vertical position) for 8 minutes.
10. With the tube on the BD IMagnet™ and using a sterile glass Pasteur pipette, carefully aspirate the supernatant (enriched fraction) and
place in a new sterile tube.
11. Remove the positive-fraction tube from the BD IMagnet™, and add 1X BD IMag™ buffer to the same volume as in Step 8. Resuspend the
positive fraction well by pipetting up and down 10 to 15 times (avoid creating bubbles), and place the tube back on the BD IMagnet™ for 6
minutes.
• For 17 x 100 mm tube: Place on the BD IMagnet™ for 8 minutes.
12. Using a new sterile Pasteur pipette, carefully aspirate the supernatant and combine with the enriched fraction from Step 10 above.
13. Repeat Steps 11 and 12. The combined enriched fraction contains CD8 T cells with no bound antibodies or magnetic particles.
14. To increase the purity of the combined enriched fraction, place the tube containing the combined enriched fraction on the BD IMagnet™ for
another 6 minutes.
• For 17 x 100 mm tube: Place on the BD IMagnet™ for 8 minutes.
15. Carefully aspirate the supernatant and place in a new sterile tube. This is the twice-enriched memory CD8+ fraction. The cells are
ready to be processed for downstream applications.
16. The positive-fraction cells remaining in the original tube can be resuspended in an appropriate buffer or culture medium for downstream
applications, including flow cytometry, if desired.
17. Samples of the total cell suspension, the positive and enriched fractions should be analyzed by flow cytometry to evaluate the efficiency of
the cell-separation procedure.
NOTES:
* Hints for successful cell preparation:
• Draw the blood into a tube containing EDTA (for example, BD Vacutainer® EDTA tube, Cat. No. 366457/ 367661).
• Remove the platelet rich plasma by centrifuging once at 220-240 × g.
• Wash 2-3 times in PBS after the density gradient separation.
• After the final wash, resuspend the cells at a relatively high concentration in 1X BD IMag™ buffer and proceed to step 3.
