The detailed Magnetic Labeling and Enrichment Protocol follows. In summary, the Biotinylated Mouse B Lymphocyte Enrichment Cocktail simultaneously stains erythrocytes and most leukocytes except the resting CD5- B lymphocytes. 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 resting CD5-B lymphocytes. 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 antibodies in the Biotinylated Mouse B Lymphocyte Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for enrichment of resting CD5- B lymphocytes from peripheral lymphoid organs.

MAGNETIC LABELING AND ENRICHMENT PROTOCOL

1. All labeling and enrichment steps may be performed in either tissue culture medium or sterile 1X BD™ IMag buffer.

- For 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 can be prepared in tissue culture medium* or 1X BD IMag 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 3. If cells are more dilute than 10 x 10^6 cells/ml, then spin down the cells and resuspend them in tissue culture medium or 1X BD IMag buffer at a concentration of 20 x 10^6 cells/ml.

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

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

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

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

8. Bring the labeling volume up to 20 to 80 x 10^6 cells/ml with tissue culture medium or 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, 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 tissue culture medium* or 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.†

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 resting CD5- B lymphocytes with no bound antibodies or magnetic particles. These cells are ready for downstream applications, or they can be further enriched by proceeding to Step 15.

14. 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.

15. To increase the purity of the combined enriched fraction by another 3% to 5% (compare middle left and middle right panels in the figure), 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.†

16. Carefully aspirate the supernatant and place in a new sterile tube. This is the twice-enriched fraction. The cells are ready to be processed for downstream applications.

17. Samples of the total cell suspension and the positive and enriched fractions should be analyzed by flow cytometry to evaluate the efficiency of the cell-separation procedure.

NOTES:Some tissue culture media contain biotin, which may interfere with the binding of the Streptavidin Particles. We recommend Dulbecco"s Minimum Essential Medium (DMEM).

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