Peripheral Blood Mononuclear Cells (PBMC) are labeled with BD IMag™ Anti-Human CD45RA Particles - DM, then placed within the magnetic field of the BD IMag™ Cell Separation Magnet. Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension to be drawn off (negative fraction). The tube is then removed from the magnetic field to resuspend the positive fraction and separated twice more to increase the purity of the positive fraction. The magnetic separation steps are diagrammed in the Separation Flow Chart. After the positive fraction is washed, it can be evaluated in downstream applications such as flow cytometry and tissue culture.
MAGNETIC LABELING AND SEPARATION PROTOCOL
1. 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), and store at 4°C.
Optional: If only CD45RA-positive T cells are desired, enrich the T lymphocytes by using the BD IMag™ Human T Lymphocyte, CD4 T Lymphocyte, or CD8 T Lymphocyte Enrichment Set - DM (Cat. No. 557874, 557939, or 557941, respectively).
2. Prepare PBMC from anti-coagulated human blood, preferably by density gradient centrifugation using Ficoll-Paque™.*
3. Count the cells, wash them with an excess volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.
4. Vortex the BD IMag™ Anti-Human CD45RA Particles - DM thoroughly, and add 50 µl of particles for every 10^7 total cells.
5. MIX THOROUGHLY. Incubate at room temperature for 30 minutes.†
6. Bring the BD IMag™-particle labeling volume up to 1 - 8 x 10^7 cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the Cell Separation Magnet.Incubate for 8 - 10 minutes.
7. With the tube on the Cell Separation Magnet, carefully aspirate off the supernatant. This supernatant contains the negative fraction.
8. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 6. Gently resuspend cells by pipetting up and down, and return the tube to the Cell Separation Magnet for another 2 - 4 minutes.
9. With the tube on the Cell Separation Magnet, carefully aspirate off the supernatant and discard.
10.Repeat Steps 8 and 9.
11.After the final wash step, resuspend the positive fraction in an appropriate buffer or medium, and proceed with desired downstream application(s).
NOTES:
* Hints for successful cell preparation:
o Draw the blood into a tube containing EDTA
o Remove the platelet rich plasma by centrifuging once at 220-240 × g.
o Wash 2-3 times in PBS after the density gradient separation.
o Remove clumps of cells and/or debris by passing the suspension through a 70-µm nylon cell strainer.
† Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.
