The detailed Magnetic Labeling and Enrichment Protocol follows. In summary, the Human Memory CD4+ T Cell Enrichment Cocktail simultaneously stains erythrocytes, platelets, and most leukocytes except the memory CD4+ helper T 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 IMag™ Cell Separation Magnet (Cat. No. 552311). Negative selection is then performed to enrich for the unlabeled memory 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 CD4+ T Cell Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for the enrichment of memory CD4+ 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 CD4+ T Cell Enrichment Cocktail at 5 μl per 1 x 10^6 cells, and incubate at room temperature for 15minutes.†

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.†

8. Bring the labeling volume up to a concentration of 20 to 80 x 10^6 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 Cell Separation Magnet (horizontal position) for 6 to 8 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 Cell Separation Magnet (vertical position for 8 minutes.)

10. With the tube on the Cell Separation Magnet 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 Cell Separation Magnet, 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 Cell Separation Magnet for 6 to 8 minutes.

· For 17 x 100 mm tube: Place on the Cell Separation Magnet 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 dendritic 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 Cell Separation Magnet for another 6 minutes.

· For 17 x 100 mm tube: Place on the Cell Separation Magnet for 8 minutes.

15. Carefully aspirate the supernatant and place in a new sterile tube. This is the twice-enriched memory CD4+ 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

· 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 4 step 3.

† Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.