BD Biosciences/Cell Separation Magnet/552311

BD IMag™ particles - DM are optimal for use with the Cell Separation Magnet. A detailed protocol for both positive and negative selection is attached. BD IMag™ particles - MSC should not be used.

For negative selection of leukocyte subsets, antibodies are selected to label the cell population(s) that are not desired. Antibody-conjugated BD IMag™ particles - DM or biotinylated antibody plus Streptavidin BD IMag™ particles - DM are used to label the cell suspension, and the labeled cells are resuspended in 1X BD IMag™ buffer. The tube(s) containing the labeled cells are then placed in the Cell Separation Magnet, where the labeled cells are attracted to the magnet, and the desired cells remain suspended in the supernatant. With the tube(s) in the Cell Separation Magnet, the supernatant is harvested to obtain the desired leukocyte population.

For positive selection of leukocyte subsets, antibodies are selected to label the desired cell population. Antibody-conjugated BD IMag™ particles -DM or biotinylated antibody plus Streptavidin BD IMag™ particles - DM are used to label the cell suspension, and the labeled cells are resuspended in 1X BD IMag™ buffer. The tube(s) containing the labeled cells are then placed in the Cell Separation Magnet, where the desired cells are attracted to the magnet. With the tube(s) in the Cell Separation Magnet, the supernatant is removed to discard the undesired leukocytes. Then the tube is removed from the Cell Separation Magnet to allow harvesting of the desired cells.

CELL SEPARATION MAGNET PROTOCOL

This protocol is suitable for separation of single-cell suspensions of 1 x 10e7 to 2 x 10e8 leukocytes in 0.5 to 10 ml.

1. Label pairs of tubes appropriately to collect positive and negative fractions and place these on ice.Dilute BD IMag™ Buffer (10X) (Cat. No. 552362) 1:10 with sterile distilled water or prepare 1X BD IMag™ buffer from scratch (Phosphate Buffered Saline, 0.5% BSA, 2 mM EDTA, and 0.09% sodium azide) and place on ice.

Steps 2 - 5 should be performed for both positive and negative selection.

2. After the cells have been labeled with BD IMag™ particles - DM, adjust the cell concentration to 2 x 10e7 cells/ml with cold 1X BD IMag™ buffer.

3. Transfer the BD IMag™-labeled cell suspension to the positive-fraction collection tube(s):

- 12 x 75 mm tube:minimum volume of 0.5 ml and maximum volume of 3.5 ml

- 17 x 100 mm tube:minimum volume of 3.0 ml and maximum volume of 10 ml

4. Place the positive-fraction tube(s) into the appropriate tube holder(s) on the Cell Separation Magnet

- 12 x 75 mm tube:leave on the Cell Separation Magnet for 6 minutes

- 17 x 100 mm tube:leave on the Cell Separation Magnet for 10 minutes

5. With the positive-fraction tube(s) on the Cell Separation Magnet, remove the supernatant (negative fraction) using a glass pasteur pipette and transfer the negative fraction into the negative-fraction tube(s). Negative cell separation has now concluded. If positive selection is desired, continue to Step 6. If positive selection is not desired, go directly to Step 11.

6. Remove the positive-fraction tube(s) from the Cell Separation Magnet, and place on ice.

To further purify the positive fraction, we recommend the addition of Steps 7 - 10.

7. Add 1 ml of cold 1X BD IMag™ buffer to each positive-fraction tube. Resuspend cells by gently pipetting or vortexing. Place the tube(s) back on the Cell Separation Magnet.

- 12 x 75 mm tube:leave on the Cell Separation Magnet for 2 minutes

- 17 x 100 mm tube:leave on the Cell Separation Magnet for 5 minutes

8. Using a new glass pasteur pipette, remove supernatant and discard.

9. Repeat Steps 6, 7, and 8 one more time.

10. Remove the positive-fraction tube(s) from the Cell Separation Magnet and place it on ice.Add 1 ml of cold 1X BD IMag™ buffer to each positive-fraction tube. Resuspend cells by gently pipetting or vortexing. Positive-cell separation has now concluded.

11. Positive and negative fractions can now be resuspended in appropriate buffers for further downstream applications, including flow cytometry.