BD IMag™ Human B Lymphocyte Enrichment Set - DM

In summary, the Biotinylated Human B Lymphocyte Enrichment Cocktail, when added to the sample, will simultaneously stain erythrocytes, platelets, and most leukocytes, but not the B lymphocytes. After washing away for any excess antibody that has not bound to cells during the staining, BD IMag™ Streptavidin Particles Plus - DM are next added to the cell suspension and will bind to the biotinylated antibodies. The tube containing this labeled cell suspension is then placed within the magnetic field of the BD IMag™ Cell Separation Magnet . Negative selection is then performed to enrich for the unlabeled B lymphocytes. Streptavidin labeled cells will migrate towards the magnet (designated as the positive fraction), leaving the unlabeled cells in suspension so they can be drawn off and retained (designated as the enriched fraction). The negative selection is repeated twice to increase the yield of the enriched fraction. If greater purity is required, further negative selection rounds may be performed on the enriched fraction. A graphical representation of the described magnetic separation procedure is diagrammed in the Enrichment Flow Chart.  The antibodies in the Biotinylated Human B Lymphocyte Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for the enrichment of B lymphocytes 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. Remove clumps of cells and/or debris by passing the suspension through a 70-µm nylon cell strainer.  Count the cells, and resuspend them in 1X BD IMag™ buffer at a concentration of 10 x 10^6 cells/ml.

4. Add the Biotinylated Human B Lymphocyte Enrichment Cocktail at 5 µl per 1 x 10^6 cells, and incubate at room temperature for 15 minutes.†

5. Wash the labeled cells with a 10X excess volume of 1X BD IMag™ buffer, centrifuge at 300 × 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. Incubate at room temperature for 30 minutes.†

8. Bring the labeling volume up to a concentration of 20-80 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, 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 by pipetting 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 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. OPTIONAL:  If the donor has a low frequency of B lymphocytes, the purity of the combined enriched fraction may be increased by another 2-8% (compare bottom middle and bottom right panels in the figure) by placing 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 (twice-enriched fraction) and place in a new sterile tube for downstream application.

17. Analyze samples of the total cell suspension, positive and enriched fractions to evaluate the efficiency of the cell-separation procedure

NOTES:

* Hints for successful cell preparation:

        o Draw blood into a tube containing EDTA

        o Remove platelet rich plasma by centrifuging once at 220-240 × g.

        o Wash 2-3 times in PBS after the density gradient separation.

         o After the final wash, resuspend the cells at a relatively high concentration in 1X BD IMag™ buffer and proceed to step 3.

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

1. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
3. BD IMag™ particles are prepared from carboxy-functionalized magnetic particles which are manufactured by Skold Technology and are licensed under US patent number 7,169,618.
4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
5. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.