Description
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.
Format
- FormatBD IMag™-DM
Suggested Companion Products
Buffer (10X) RUO 100mLCat No: 552362
Cell Separation Magnet RUO Cat No: 552311
Human T Lymphocyte Enrichment Set-DM RUO Cat No: 557874
APC Mouse Anti-Human CD3 UCHT1 (also known as UCHT-1; UCHT 1)RUO 100TestsCat No: 555335
FITC Mouse Anti-Human CD45RO UCHL1RUO 100TestsCat No: 555492
Human CD4 T Lymphocyte Enrichment Set-DM RUO Cat No: 557939
Human CD8 T Lymphocyte Enrichment Set-DM RUO Cat No: 557941
Propidium Iodide Staining Solution RUO 2mLCat No: 556463
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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.
Product Notices
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
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.
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