BD IMag™ Anti-Mouse IgG1 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 0.09% sodium azide.*

2. Prepare a single-cell suspension from the lymphoid tissue of interest or prepare peripheral blood mononuclear cells (PBMC) from anti-coagulated blood, preferably by density gradient centrifugation using the appropriate density Ficoll-Paque™ 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. Add the mouse IgG1 antibody (or cocktail of mouse IgG1 antibodies) at the appropriate concentration, and incubate on ice for 15 minutes.†

5. Wash the labeled cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate ALL the supernatant. For depletions, proceed with Step 6. For positive selections, proceed with Step 17.

Depletions:

6. Vortex the BD IMag™ Anti-Mouse IgG1 Magnetic Particles - DM thoroughly, and add 50 µl of particles for every 1 x 10e7 total cells.

7. MIX THOROUGHLY. Refrigerate rat or mouse leukocytes for 30 minutes at 6°C -12°C. Incubate human PBMC at room temperature for 30 minutes.†

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

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 6-8 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 glass Pasteur pipette, carefully aspirate the supernatant (depleted fraction) and place in a new tube.

11. 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.

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

13. Repeat Steps 11 and 12. 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 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.

15. 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.

16. Carefully aspirate the supernatant (the final depleted fraction) and place in a new tube. The cells are ready for downstream applications.  

Positive Selections:

17. Vortex the BD IMag™ Anti-Mouse IgG1 Magnetic Particles - DM thoroughly, and add 10-50 µl of particles for every 1 x 10e7 total cells.

18. MIX THOROUGHLY. Refrigerate rat or mouse leukocytes for 30 minutes at 6°C -12°C. Incubate human PBMC at room temperature for 30 minutes.†

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

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

21. With the tube on the Cell Separation Magnet, carefully aspirate the supernatant. This supernatant is considered the Negative Fraction.

22. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 19. Gently resuspend the cells by pipetting up and down, and return the tube to the Cell Separation Magnet for another 2-4 minutes.

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

24. Repeat Steps 22 and 23.

25. 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 rat 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. 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.
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. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.