In summary, the Human Monocyte Enrichment Cocktail, when added to the sample, will simultaneously stain erythrocytes, and most leukocytes which will result in an enriched monocyte cell population. Addition of the optional biotin mouse anti-human CD41a antibody will allow for the removal of platelets. After washing away for any 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 the 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 monocytes. Streptavidin labeled cells migrate toward 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, negative selection may be performed on the enriched fraction. A graphical representation of the described magnetic separation procedure is diagrammed in the Enrichment Flow Chart. The biotinylated antibodies in the Human Monocyte Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for the enrichment of monocytes 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 10e6 cells/ml.
4. Add the Human Monocyte Enrichment Cocktail at 5 µl per 1 x 10e6 cells, and incubate at room temperature for 15 minutes.†
Optional: Add the Biotin Mouse anti-human CD41a at 5 µl per 1 x 10e6 cells
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 resuspend the cell pellet in 5 µl of particles per 1 x 10e6 cells.
7. MIX THOROUGHLY. Incubate at room temperature for 30 minutes.†
8. Bring the labeling volume up to 20-80 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 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 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 monocytes with no bound antibodies or magnetic particles.
14. To increase the purity of the combined enriched fraction by another 5% or more (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 10 minutes.
-For 17 x 100 mm tube: Place on the Cell Separation Magnet for 10 minutes.
15. 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.
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 and 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 step 3.
† Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.