The detailed Magnetic Labeling and Enrichment Protocol follows. In summary, the Biotinylated Mouse Dendritic Cell Enrichment Cocktail simultaneously stains erythrocytes and most leukocytes except the DC. 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 Cell Separation Magnet (Cat. No. 552311). Negative selection is then performed to enrich for the unlabeled DC. 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 once to increase the yield of the enriched fraction. For greater purity, negative selection is performed on the enriched fraction. For clarification of the procedure, the magnetic separation steps are diagrammed in the Enrichment Flow Chart. The enriched and positive fractions can be evaluated in downstream applications such as flow cytometry and tissue culture. The antibodies in the Biotinylated Mouse Dendritic Cell Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for enrichment of DC from peripheral lymphoid organs.

MAGNETIC LABELING AND ENRICHMENT PROTOCOL

1. Prepare sterile buffers and place on ice.

- Cell-staining buffer: Phosphate Buffered Saline supplemented with 3% heat-inactivated fetal calf serum and 0.1% sodium azide

- 1X BD IMag buffer: Dilute BD IMag Buffer (10X) (Cat. No. 552362) 1:10 with sterile distilled water or prepare Phosphate Buffered Saline supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide.

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. Cell suspensions should be prepared in cell-staining buffer.

3. Count the cells. If the concentration is between 10 x 10^6 and 20 x 10^6 cells/ml, then proceed to Step 4. If cells are more dilute than 10 x 10^6 cells/ml, then spin down the cells and resuspend them in cell-staining buffer at a concentration of 20 x 10^6 cells/ml.

4. OPTIONAL: Add BD Mouse Fc Block Purified Anti-Mouse CD16/CD32 mAb 2.4G2 (Cat. No. 553141/553142) at 0.25 µg per 1 x 10^6 cells, and incubate on ice for 15 minutes.

5. Add the Bioinylated Mouse Dendritic Cell Enrichment Cocktail at 5 µl per 1 x 10^6 cells, and incubate on ice for 15 minutes.†

6. Wash the labeled cells with a 10X excess volume of 1X BD IMag buffer, centrifuge at 300 x g for 7 minutes, and carefully aspirate ALL the supernatant.

7. Vortex the BD IMag Streptavidin Particles Plus - DM thoroughly, and add 5 µl of particles for every 1 x 10^6 total cells.

8. MIX THOROUGHLY. Refrigerate for 30 minutes at 6°C - 12°C.†

9. Bring the labeling volume up to a concentration of 20-80 x 10^6 cells/ml with 1X BD IMag buffer.

10. 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, 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.

11. 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.

12. 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, 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.

13. Using a new sterile Pasteur pipette, carefully aspirate the supernatant and combine with the enriched fraction from Step 10 above.

14. Place the tube containing the combined enriched fraction on the Cell Separation Magnet for another 6 to 8 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 twice-enriched fraction contains DC with no bound antibodies or magnetic particles.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 unseparated cell suspension and the positive and enriched fractions should be analyzed by flow cytometry to evaluate the efficiency of the cell-separation procedure.

NOTES:

- The use of BD Mouse Fc Block™ Purified Anti-Mouse CD16/CD32 mAb 2.4G2 in step 4 increases the yield by 15 - 25% while decreasing the purity of the DC by 7 - 15%. Please note that this results in enriched DC which may have purified anti-mouse CD16/CD32 mAb bound to their surface, which may affect the function of those DC.

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