Skip to main content Skip to navigation
Anti-Human CD45RO Particles - DM
Anti-Human CD45RO Particles - DM

Positive selection of CD45RO+ leukocytes and CD45RO+ T lymphocytes from PBMC from two different donors. Leukocytes from the first donor (top panels) were labeled with BD™ IMag Anti-Human CD45RO Particles - DM (Cat. No. 557986), separated using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), and the negative (CD45RO-) and positive (CD45RO+) fractions were collected. T lymphocytes were enriched from the second donor's PBMC (bottom panels) using the BD™ IMag Human T Lymphocyte Enrichment Set (Cat. No. 557874) and then the CD45RO+ cells were collected. Refer to the Positive Selection Flow Chart to identify the separated cell populations represented. For flow cytometric analysis, fresh PBMC (left panels), the negative fraction (top middle panel), the positive fraction (top right panel), enriched T lymphocytes (bottom middle panel), and CD45RO+ T lymphocytes (bottom right panel) were stained with FITC Mouse Anti-human CD45RO (Cat. No. 555492) and APC Mouse Anti-human CD3 (Cat. No. 555335). Non-viable cells were excluded from analysis by staining with Propidium Iodide Staining Solution (Cat. No. 556463). The percentages of CD45RO+ cells (top three panels) or CD45RO+ T lymphocytes (bottom panels) in each sample is given. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Positive selection of CD45RO+ leukocytes and CD45RO+ T lymphocytes from PBMC from two different donors. Leukocytes from the first donor (top panels) were labeled with BD™ IMag Anti-Human CD45RO Particles - DM (Cat. No. 557986), separated using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), and the negative (CD45RO-) and positive (CD45RO+) fractions were collected. T lymphocytes were enriched from the second donor's PBMC (bottom panels) using the BD™ IMag Human T Lymphocyte Enrichment Set (Cat. No. 557874) and then the CD45RO+ cells were collected. Refer to the Positive Selection Flow Chart to identify the separated cell populations represented. For flow cytometric analysis, fresh PBMC (left panels), the negative fraction (top middle panel), the positive fraction (top right panel), enriched T lymphocytes (bottom middle panel), and CD45RO+ T lymphocytes (bottom right panel) were stained with FITC Mouse Anti-human CD45RO (Cat. No. 555492) and APC Mouse Anti-human CD3 (Cat. No. 555335). Non-viable cells were excluded from analysis by staining with Propidium Iodide Staining Solution (Cat. No. 556463). The percentages of CD45RO+ cells (top three panels) or CD45RO+ T lymphocytes (bottom panels) in each sample is given. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
Down Arrow Up Arrow


BD IMag™
CD45R; PTPRC; LCA; Leukocyte common antigen; GP180; LY5; T200
Human (QC Testing)
Mouse BALB/c IgG2a, κ
Human IL-2-dependent T-cell line
Cell separation (Routinely Tested)
IV N31
5788
AB_398641
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

Peripheral Blood Mononuclear Cells (PBMC) are labeled with BD IMag™ Anti-Human CD45RO Particles - DM according to the following Protocol. This labeled cell suspension is then placed within the magnetic field of the BD IMag™ Cell Separation Magnet. 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, it can be further evaluated in downstream applications such as flow cytometry and tissue culture.

MAGNETIC LABELING AND SEPARATION PROTOCOL

1. 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), and store at 4°C.

Optional: If only CD45RO-positive T cells are desired, enrich the T lymphocytes by using the BD IMag™ Human T Lymphocyte, CD4 T Lymphocyte, or CD8 T Lymphocyte Enrichment Set - DM (Cat. No. 557874, 557939, or 557941, respectively).

2. Prepare PBMC from anti-coagulated human blood, preferably by density gradient centrifugation using Ficoll-Paque™.*

3. Count the cells, wash them with an excess volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.

4. Vortex the BD IMag™ Anti-Human CD45RO Particles - DM thoroughly, and add 50 µl of particles for every 10^7 total cells.

5. MIX THOROUGHLY. Incubate at room temperature for 30 minutes.†

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

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

8. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 6. Gently resuspend cells by pipetting up and down, and return the tube to the Cell Separation Magnet for another 2 - 4 minutes.

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

10. Repeat Steps 8 and 9.

11. After the final wash step, resuspend the positive fraction in an appropriate buffer or medium, and proceed with desired downstream application(s).

NOTES:

* Hints for successful cell preparation:

-Draw the blood into a tube containing EDTA

-Remove the platelet rich plasma by centrifuging once at 220-240 × g.

-Wash 2-3 times in PBS after the density gradient separation.

-Remove clumps of cells and/or debris by passing the suspension through a 70-µm nylon cell strainer.

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

Product Notices

  1. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. 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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
557986 Rev. 2
Antibody Details
Down Arrow Up Arrow
UCHL1

BD IMag™ Anti-Human CD45RO Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. These particles are optimized for the positive selection of CD45RO-bearing leukocytes using the BD IMag™  Cell Separation Magnet (Cat. No. 552311). CD45RO is the 180 kDa isoform of leukocyte common antigen that is not encoded by exon A, B, or C and found on most thymocytes, activated T lymphocytes, granulocytes, and monocytes and on a major subset of peripheral T lymphocytes. CD45RO and CD45RA expression defines complementary, predominantly non-overlapping populations of T cells in peripheral blood; and it is generally accepted that naive T cells are CD45RO- CD45RA+, while memory T cells are CD45RO+ CD45RA-. To specifically enrich CD45RO-expressing memory T lymphocytes, we recommend first depleting the erythrocytes, platelets, and non-T leukocytes, by using the appropriate BD IMag™ human T lymphocyte enrichment set, followed by positive selection of the CD45RO+ population.

557986 Rev. 2
Format Details
Down Arrow Up Arrow
BD IMag DM
BD IMag DM
557986 Rev.2
Citations & References
Down Arrow Up Arrow

Development References (6)

  1. 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.
  2. Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
  3. Picker LJ, Treer JR, Ferguson-Darnell B, Collins PA, Buck D, Terstappen LW. Control of lymphocyte recirculation in man. I. Differential regulation of the peripheral lymph node homing receptor L-selectin on T cells during the virgin to memory cell transition. J Immunol. 1993; 150(3):1105-1121. (Biology). View Reference
  4. Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995.
  5. Schwinzer R. Cluster Report: CD45/CD45R. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:628-634.
  6. Streuli M, Morimoto C, Schrieber M, Schlossman SF, Saito H. Characterization of CD45 and CD45R monoclonal antibodies using transfected mouse cell lines that express individual human leukocyte common antigens. J Immunol. 1988; 141(11):3910-3914. (Biology). View Reference
View All (6) View Less
557986 Rev. 2

Please refer to Support Documents for Quality Certificates


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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.