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Anti-Mouse CD45R/B220 Magnetic Particles - DM
Anti-Mouse CD45R/B220 Magnetic Particles - DM
Positive selection and depletion of mouse CD45R/B220-positive splenocytes. Leukocytes were labeled with BD IMag™ anti-mouse CD45R/B220 Particles - DM (Cat. No. 551513) as described in the protocol. After labeling, the cells were separated using the BD IMag™Cell Separation Magnet (Cat. No. 552311), and the negative (CD45R/B220-) and positive (CD45R/B220+) fractions were collected. Please refer to the Separation Flow Chart to identify the separated cell populations represented in this figure. For flow cytometric analysis, fresh splenocytes (left panel), the negative fraction (middle panel), and the positive fraction (right panel) were stained with FITC Rat Anti-Mouse CD45R/B220  (Cat. No. 553087) and PE Rat Anti-mouse CD13 (Cat. No. 557399). The percent CD45R/B220+/CD19+ cells in each sample is given in the upper right corner.
Positive selection and depletion of mouse CD45R/B220-positive splenocytes. Leukocytes were labeled with BD IMag™ anti-mouse CD45R/B220 Particles - DM (Cat. No. 551513) as described in the protocol. After labeling, the cells were separated using the BD IMag™Cell Separation Magnet (Cat. No. 552311), and the negative (CD45R/B220-) and positive (CD45R/B220+) fractions were collected. Please refer to the Separation Flow Chart to identify the separated cell populations represented in this figure. For flow cytometric analysis, fresh splenocytes (left panel), the negative fraction (middle panel), and the positive fraction (right panel) were stained with FITC Rat Anti-Mouse CD45R/B220  (Cat. No. 553087) and PE Rat Anti-mouse CD13 (Cat. No. 557399). The percent CD45R/B220+/CD19+ cells in each sample is given in the upper right corner.
Product Details
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BD IMag™
B220; Ly-5; CD45R; LCA; Ptprc; Protein tyrosine phosphatase receptor type C
Mouse (QC Testing)
Rat IgG2a, κ
Mouse Abelson Leukemia Virus-Induced pre-B tumor cells
Cell separation (Routinely Tested)
AB_10051958
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C. 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

Leukocytes are labeled with BD IMag™ anti-mouse CD45R/B220 Particles - DM according to the Magnetic Labeling Protocol. This labeled cell suspension is then placed within the magnetic field of the BD IMag™ Cell Separation Magnet (Cat. No. 552311). Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off (negative fraction). The tube is then removed from the magnetic field for resuspension of the positive fraction. The separation is repeated twice 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, the small size of the magnetic particles allows the positive fraction to be further evaluated in downstream applications such as flow cytometry.

MAGNETIC LABELING PROTOCOL

1. Prepare a single-cell suspension from the lymphoid tissue of interest according to standard laboratory procedures. Remove clumps of cells and/or debris by passing the suspension through a 70-µm nylon cell strainer.

2. Dilute BD IMag™ Buffer (10X) (Cat. No. 552362) 1:10 with sterile distilled water or prepare 1X BD IMag™ buffer by supplementing Phosphate Buffered Saline with 0.5% BSA, 2mM EDTA, and 0.09% sodium azide. Place on ice.

Although our experience indicates that use of Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 (Cat. No. 553141/553142) is not required for optimal cell separation, some laboratories may want to use it in their studies.

If adding Mouse BD Fc Block™, proceed to Step 3.If not adding Mouse BD Fc Block™, proceed to Step 4.

3. Add Mouse BD Fc Block™ at 0.25 µg/10e6 cells, and incubate on ice for 15 minutes.

4. Wash cells with at least an equal volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.

5. Vortex the BD IMag™ anti-mouse CD45R/B220 Particles - DM thoroughly, and add 50 µl of particles for every 10e7 total cells.

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

7. Bring the BD IMag-particle labeling volume up to 1-8 x 10e7 cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the Cell Separation Magnet. Incubate at room temperature for 6-8 minutes.

8. With the tube on the Cell Separation Magnet, carefully aspirate off the supernatant. This supernatant contains the negative fraction.

9. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 7. Gently resuspend cells by pipetting briefly, and return the tube to the Cell Separation Magnet for another 2-4 minutes.

10. With the tube on the Cell Separation Magnet, carefully aspirate off the supernatant and discard.

11. Repeat steps 9 and 10.

12. After the final wash step, resuspend the positive fraction in an appropriate buffer and at an appropriate concentration for further analysis.

Note: Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.

Product Notices

  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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
551513 Rev. 2
Antibody Details
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RA3-6B2

BD IMag™ anti-mouse CD45R/B220 Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. These particles are optimized for the positive selection or depletion of CD45R/B220-bearing leukocytes using the BD IMag™ Cell Separation Magnet. CD45R/B220 is reportedly expressed on B lymphocytes at all stages from pro-B through mature and activated B cell. It is also has been reported to be found on abnormal T cells involved in the lymphadenopathy of lpr/lpr and gld/gld mutant mice, on lytically active subsets of lymphokine-activated killer cells (NK cells and non-MHC-restricted CTL), on apoptotic T lymphocytes of mice injected with bacterial superantigen, on a population of NK-cell precursors in the bone marrow, and on B-lymphocyte, T-lymphocyte, and macrophage progenitors in fetal liver. The CD45R/B220 antigen is reportedly not on hematopoietic stem cells, plasma cells, resting T lymphocytes, or MHC-restricted CTL.

551513 Rev. 2
Format Details
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BD IMag DM
BD IMag DM
551513 Rev.2
Citations & References
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Development References (10)

  1. Ballas ZK, Rasmussen W. Lymphokine-activated killer cells. VII. IL-4 induces an NK1.1+CD8 alpha+beta- TCR-alpha beta B220+ lymphokine-activated killer subset. J Immunol. 1993; 150(1):17-30. (Biology). View Reference
  2. Coffman RL. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B cell development. Immunol Rev. 1982; 69:5-23. (Biology). View Reference
  3. Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J Exp Med. 1991; 173(5):1213-1225. (Biology). View Reference
  4. Hathcock KS, Hirano H, Murakami S, Hodes RJ. CD45 expression by B cells. Expression of different CD45 isoforms by subpopulations of activated B cells. J Immunol. 1992; 149(7):2286-2294. (Biology). View Reference
  5. Johnson P, Maiti A, Ng DHW. CD45: A family of leukocyte-specific cell surface glycoproteins. In: Herzenberg LA, Weir DM, Herzenberg LA, Blackwell C , ed. Weir's Handbook of Experimental Immunology, Vol 2. Cambridge: Blackwell Science; 1997:62.1-62.16.
  6. Kobata T, Takasaki K, Asahara H, et al. Apoptosis with FasL+ cell infiltration in the periphery and thymus of corrected autoimmune mice. Immunology. 1997; 92(2):206-213. (Biology). View Reference
  7. Laouar Y, Ezine S. In vivo CD4+ lymph node T cells from lpr mice generate CD4-CD8-B220+TCR-beta low cells. J Immunol. 1994; 153(9):3948-3955. (Biology). View Reference
  8. Renno T, Hahne M, Tschopp J, MacDonald HR. Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand. J Exp Med. 1996; 183(2):431-437. (Biology). View Reference
  9. Rolink A, ten Boekel E, Melchers F, Fearon DT, Krop I, Andersson J. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J Exp Med. 1996; 183(1):187-194. (Biology). View Reference
  10. Sagara S, Sugaya K, Tokoro Y, et al. B220 expression by T lymphoid progenitor cells in mouse fetal liver. J Immunol. 1997; 158(2):666-676. (Biology). View Reference
View All (10) View Less
551513 Rev. 2

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