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Anti-Rat Ig, κ Light Chain Particles - DM
Anti-Rat Ig, κ Light Chain Particles - DM

Depletion of T-, NK-, B-, and myeloid-lineage cells from mouse bone marrow (top panels). BALB/c bone marrow cells (BM) were stained with FITC Rat Anti-Mouse CD3 Molecular Complex (Cat. No. 555274), CD11b (Cat. No. 553310/557396/561688), CD45R/B220 (Cat. No. 553087/553088), and Ly-6G and Ly-6C (Gr-1) (Cat. No. 553126/553127), then labeled with BD  IMag™ Anti-Rat Ig, κ Light Chain Particles - DM (Cat. No. 558002). Cells were separated using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), and the negative and positive fractions were collected as described in the following depletion protocol. Refer to the depletion flow chart to identify the separated cell populations represented in this figure. Nonviable cells were eliminated from analysis by staining with Propidium Iodide Staining Solution (Cat. No. 556463), and all viable cells are displayed. The percentage of non-stained cells, which include the erythroid lineage and non-lineage-committed leukocytes, in each sample are given. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Positive selection of CD8a-positive T lymphocytes from mouse spleen (bottom panels). BALB/c splenocytes were stained with FITC Rat Anti-Mouse CD8a (Cat. No. 553030/553031) and then labeled with BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM. Cells were separated using the BD IMag™ Cell Separation Magnet, and the negative and positive fractions were collected as described in the positive selection protocol. Refer to the positive selection flow chart to identify the separated cell populations represented in this figure. Nonviable cells were eliminated from analysis by staining with Propidium Iodide Staining Solution, and all viable cells are displayed. The percentage of CD8a+ cells in each sample are given. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Anti-Rat Ig, κ Light Chain Particles - DM

Optimal concentrations of BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM for positive selection with some rat Ig κ monoclonal antibodies to mouse leukocyte antigens.

Depletion of T-, NK-, B-, and myeloid-lineage cells from mouse bone marrow (top panels). BALB/c bone marrow cells (BM) were stained with FITC Rat Anti-Mouse CD3 Molecular Complex (Cat. No. 555274), CD11b (Cat. No. 553310/557396/561688), CD45R/B220 (Cat. No. 553087/553088), and Ly-6G and Ly-6C (Gr-1) (Cat. No. 553126/553127), then labeled with BD  IMag™ Anti-Rat Ig, κ Light Chain Particles - DM (Cat. No. 558002). Cells were separated using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), and the negative and positive fractions were collected as described in the following depletion protocol. Refer to the depletion flow chart to identify the separated cell populations represented in this figure. Nonviable cells were eliminated from analysis by staining with Propidium Iodide Staining Solution (Cat. No. 556463), and all viable cells are displayed. The percentage of non-stained cells, which include the erythroid lineage and non-lineage-committed leukocytes, in each sample are given. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Positive selection of CD8a-positive T lymphocytes from mouse spleen (bottom panels). BALB/c splenocytes were stained with FITC Rat Anti-Mouse CD8a (Cat. No. 553030/553031) and then labeled with BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM. Cells were separated using the BD IMag™ Cell Separation Magnet, and the negative and positive fractions were collected as described in the positive selection protocol. Refer to the positive selection flow chart to identify the separated cell populations represented in this figure. Nonviable cells were eliminated from analysis by staining with Propidium Iodide Staining Solution, and all viable cells are displayed. The percentage of CD8a+ cells in each sample are given. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Optimal concentrations of BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM for positive selection with some rat Ig κ monoclonal antibodies to mouse leukocyte antigens.

Product Details
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BD IMag™
Mouse IgG1
Pooled rat Ig
Cell separation (Routinely Tested)
AB_398644
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Antibody or streptavidin was conjugated to the magnetic particles under optimum conditions, and unconjugated antibody/streptavidin was removed.

Recommended Assay Procedures

A detailed Magnetic Labeling and Separation Protocol follows. In brief, cells are labeled with a rat Ig κ antibody that recognizes the subpopulation of interest. After washing away excess antibody, BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM are added to the cell suspension and bind the rat Ig antibody on the cells. The tube containing this labeled cell suspension is then placed within the magnetic field of the Cell Separation Magnet. Positive selection or depletion is then performed. Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off (depleted or negative fraction). The tube is then removed from the magnetic field for resuspension of the positive fraction. The selections are repeated twice to increase the purity of the positive fraction and the yield of the depleted fraction. The magnetic separation steps are diagrammed in the accompanying depletion and positive selection flow charts. The small size of the BD IMag™ particles allows the positive fraction to be further evaluated in downstream applications such as flow cytometry.

MAGNETIC LABELING AND SEPARATION PROTOCOL

1. Prepare 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 prepare Phosphate Buffered Saline, supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide.*

2. Aseptically 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-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 rat Ig antibody (or cocktail of rat Ig κ 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-Rat Ig, κ Light Chain Particles - DM thoroughly, and add 50 µl of particles for every 1 x 10e7 total cells.

7. MIX THOROUGHLY. Refrigerate for 30 minutes at 6°C - 12°C.**

8. Bring the labeling volume up to a concentration of 2 to 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 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 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 to 15 times and place back on the Cell Separation Magnet for 6 to 8 minuets.

        · 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 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 to 8 minutes.

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

16. Carefully aspirate the supernatant and place in a new tube. This is the final depleted fraction. The cells are ready to be processed for downstream applications.

Positive Selections:

17. Vortex the BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM thoroughly, and add 10 to 50 µl of particles for every 1 x 10e7 total cells. The amount of particles to add will vary depending on how many cells one is targeting and the cell-surface density of the antigen. Please refer to the table below for some common examples.

18. MIX THOROUGHLY. Refrigerate for 30 minutes at 6°C - 12°C.

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

20. Immediately place the tube onto the Cell Separation Magnet and incubate for 6 to 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 to 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 mouse leukocytes, tissue culture medium usually results in a slight increase in viability and recovery, when compared to IMag buffer, without reducing cell purity. We recommend that researchers run a trial comparison of media to buffer to make sure that there are no adverse effects.

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

Product Notices

  1. 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.
  2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. 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.
  4. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558002 Rev. 3
Antibody Details
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MRK-1

BD IMag™ Anti-Rat Ig, κ Light Chain Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. These particles are optimized for the positive selection or depletion of leukocyte subpopulations using the BD IMag™ Cell Separation Magnet (Cat. No. 552311). The MRK-1 antibody reacts specifically with rat immunoglobulins bearing κ light chain (rat Ig κ). It does not react with λ light chain or heavy chains.

558002 Rev. 3
Format Details
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BD IMag DM
BD IMag DM
558002 Rev.3

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.