BD IMag™ Anti- R-Phycoerythrin (PE) Magnetic Particles - DM

MAGNETIC LABELING AND SEPARATION PROTOCOL

1.   Prepare the following buffers and place on ice:

      a. Cell-staining buffer: Phosphate Buffered Saline, 3% heat inactivated fetal calf serum, 0.1% sodium azide.

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

2.   Prepare a single-cell suspension from the lymphoid tissue of interest or prepare PBMC from anti-coagulated blood, preferably by density gradient centrifugation using the appropriate density Ficoll-Hypaque™ solution. Remove clumps of cells and/or debris by passing the suspended cells through a 70-µm nylon cell strainer.

3.   Count the cells, and resuspend them in cell-staining buffer at a concentration of 2 x 10e7 cells/ml.

4.   Optional: If appropriate, add BD Mouse Fc Block™ Purified Anti-Mouse CD16/CD32 (Cat. No. 553141) or BD Rat Fc Block™ Purified Anti-Rat CD32 (Cat. No. 550270) at 0.25 µg/10e6 cells, and incubate on ice for 15 minutes.

5.   Add the PE-conjugated antibody (or antibody cocktail) at the appropriate concentration, and incubate on ice for 15 minutes. †

6.   Wash the labeled cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate ALL the supernatant. For depletions, proceed with Step 7. For positive selections, proceed with Step 18.

Depletions:

7.   Vortex the Anti- R-Phycoerythrin (PE) Magnetic Particles - DM thoroughly, and add 50 µl of particles per 1x10e7 total cells.

8.   MIX THOROUGHLY. Incubate mouse/ rat leukocytes for 30 minutes at 6°C-12°C and human PBMC at room temperature for 30 minutes. †

9.   Bring the labeling volume up to a concentration of 2-8 x 10e7 cells/ml with 1X BD IMag™ buffer or culture medium.*

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 6-8 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, use a glass Pasteur pipette to aspirate the supernatant (depleted fraction) into a new tube.

12. Remove the positive fraction tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer (or medium) to the same volume as in Step 9. Resuspend the positive fraction well by pipetting up and down 10-15 times and place back on the Cell Separation Magnet for 6-8 minutes.

        - 17 x 100 mm tube: Place on the Cell Separation Magnet for 8 minutes.

13. Using a new Pasteur pipette, carefully aspirate the supernatant and combine with the depleted fraction from Step 11 above.

14. Repeat Steps 12 and 13. The combined depleted fraction contains cells with no bound antibodies or magnetic particles. These cells are ready for downstream applications, or they can be further enriched by proceeding to Step 16.

15. 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.

16. To increase the purity of the combined depleted fraction, place the tube on the Cell Separation Magnet for another 6-8 minutes.

        - 17 x 100 mm tube: Place on the Cell Separation Magnet for 8 minutes.

17. Carefully aspirate the supernatant (final depleted fraction) into a new tube. The cells are ready to be processed for downstream applications.

Positive Selections:

18. Vortex the Anti- R-Phycoerythrin (PE) Magnetic Particles - DM thoroughly, and add 10-50 µl of particles per1 x 10e7 total cells.  

Note:  The amount of particles to add may vary depending on how many cells one is targeting and the cell-surface density of the antigen. Please refer to Table 1 for some common examples.

19. MIX THOROUGHLY. Incubate mouse/ rat leukocytes for 30 minutes at 6°C - 12°C and human PBMC at room temperature for 30 minutes. †

20. Bring the labeling volume up to a concentration of 2-8 x 10e7 cells/ml with 1X BD IMag™ buffer.

21. Immediately place the tube onto the Cell Separation Magnet and incubate for 6-8 minutes.

22. With the tube on the Cell Separation Magnet, carefully aspirate the supernatant. This supernatant is the negative fraction.

23. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 20. Gently resuspend the cells by pipetting up and down, and return the tube to the Cell Separation Magnet for another 2-4 minutes.

24. With the tube on the Cell Separation Magnet, carefully remove the supernatant.

25. Repeat Steps 23 and 24.

26. After the final wash step, remove the tube from the Cell Separation Magnet.  Resuspend the Positive Fraction in an appropriate buffer or culture medium, and proceed with desired downstream application(s), including flow cytometry.

NOTES: * For depletion of mouse leukocytes, tissue culture medium usually results in a slight increase in viability and recovery, when compared to IMag buffer, without reducing cell purity. Because applications can vary, researchers are encouraged to run a trial comparison of culture media and IMag buffer to demonstrate that there are no adverse effects.

† Avoid non-specific labeling by working quickly and adhering to recommended incubation times.

1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
4. 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.
5. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.