Description
The BD IMag™ Human T Lymphocyte Enrichment Set - DM is used for the negative selection of T lymphocytes from peripheral blood. The Biotin Human T Lymphocyte Enrichment Cocktail contains monoclonal antibodies that recognize antigens expressed on erythrocytes, platelets, and peripheral leukocytes that are not T lymphocytes. The BD IMag™ Streptavidin Particles Plus - DM are magnetic nanoparticles that have streptavidin covalently conjugated to their surfaces. With these two components, the BD IMag™ Human T Lymphocyte Enrichment Set - DM avoids the inadvertent activation of the enriched T lymphocytes by using reagents that do not directly bind to those T cells. This Enrichment Set has been optimized for use with the BD IMag™ Cell Separation Magnet, and it contains sufficient reagents to label 10^9 peripheral blood mononuclear cells (PBMC).
The T Lymphocyte Enrichment Cocktail component is comprised of the following biotin-conjugated monoclonal antibodies:
Anti-human CD11b/Mac-1 (CR3), clone ICRF44
Anti-human CD16, clone 3G8
Anti-human CD19, clone HIB19
Anti-human CD36, clone CB38 (NL07)
Anti-human CD41a, clone HIP8
Anti-human CD56, clone B159
Anti-human CD235a (Glycophorin A), clone GA-R2 (HIR2)
Components
Part No. | Description | Size |
---|---|---|
51-9000810 | Streptavidin Particles Plus - DM | NA |
51-9002184 | Biotin Human T Lymphocyte Enrichment Cocktail | NA |
Format
- FormatBD IMag™-DM
Other Formats→
- Purified
Suggested Companion Products
APC Mouse Anti-Human CD3 UCHT1 (also known as UCHT-1; UCHT 1)RUO 100TestsCat No: 555335
Cell Separation Magnet RUO Cat No: 552311
PE Mouse Anti-Human CD11b ICRF44 (also known as 44)RUO 100TestsCat No: 555388
PE Mouse Anti-Human CD16 3G8RUO 100TestsCat No: 555407
PE Mouse Anti-Human CD19 HIB19RUO 100TestsCat No: 555413
PE Mouse Anti-Human CD36 CB38 (also known as NL07)RUO 100TestsCat No: 555455
PE Mouse Anti-Human CD56 B159RUO 100TestsCat No: 555516
PE Mouse Anti-Human CD235a GA-R2 (HIR2)RUO 0.1mgCat No: 555570
Buffer (10X) RUO 100mLCat No: 552362
Resources & Tools | ||||||
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SpectrumViewer | Download TDS | Regulatory Document Website |
Preparation and Storage
Store undiluted at 4°C.The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.The antibody was conjugated with biotin under optimum conditions, and unreacted biotin was removed.Antibody or streptavidin was conjugated to the magnetic particles under optimum conditions, and unconjugated antibody/streptavidin was removed.
Product Notices
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Ficoll-Paque is a trademark of Amersham Biosciences Limited.
- 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/us/s/resources for technical protocols.
The detailed Magnetic Labeling and Enrichment Protocol follows. In summary, the Biotinylated Human T Lymphocyte Enrichment Cocktail simultaneously stains erythrocytes, platelets, and most leukocytes except the T lymphocytes. After washing away excess antibody, BD IMag™ Streptavidin Particles Plus - DM are added to the cell suspension and bind the cells bearing the biotinylated antibodies. The tube containing this labeled cell suspension is then placed within the magnetic field of the BD IMag™Cell Separation Magnet (Cat. No. 552311). Negative selection is then performed to enrich for the unlabeled T cells. Labeled cells migrate toward the magnet (positive fraction), leaving the unlabeled cells in suspension so they can be drawn off and retained (enriched fraction). The negative selection is repeated twice to increase the yield of the enriched fraction. If greater purity is required, negative selection may be performed on the enriched fraction. For clarification of the procedure, the magnetic separation steps are diagrammed in the Enrichment Flow Chart. The enriched (and positive) fractions can be evaluated in downstream applications such as flow cytometry and tissue culture. The antibodies in the Biotinylated Human T Lymphocyte Enrichment Cocktail have been optimized and pre-diluted to provide maximum efficiency for enrichment of T lymphocytes from PBMC.
MAGNETIC LABELING AND ENRICHMENT PROTOCOL
1.Prepare 1X BD IMag™ buffer: Dilute BD IMag™ Buffer (10X) (Cat. No. 552362) 1:10 with sterile distilled water or prepare Phosphate Buffered Saline (PBS) supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide.
2.Prepare PBMC from anti-coagulated human blood, preferably by density gradient centrifugation using Ficoll-Paque™.*
3.Remove clumps of cells and/or debris by passing the suspension through a 70-µm nylon cell strainer. Count the cells, and resuspend them in 1X BD IMag™ buffer at a concentration of 10 x 10^6 cells/ml.
4.Add the Biotinylated Human T Lymphocyte Enrichment Cocktail at 5 µl/1 x 10^6 cells, and incubate at room temperature for 15 minutes.†
5.Wash the labeled cells with a 10X excess volume of 1X BD IMag™ buffer, centrifuge at 300 × g for 7 minutes, and carefully aspirate ALL the supernatant.
6.Vortex the BD IMag™ Streptavidin Particles Plus - DM thoroughly, and add 5 µl of particles for every 1 x 10^6 total cells.
7.MIX THOROUGHLY. Incubate at room temperature for 30 minutes.†
8.Bring the labeling volume up to a concentration of 20-80 x 10^6 cells/ml with 1X BD IMag™ buffer.
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 sterile glass Pasteur pipette, carefully aspirate the supernatant (enriched fraction) and place in a new sterile tube.
11.Remove the positive-fraction tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 8. Resuspend the positive fraction well by pipetting up and down 10 to 15 times, and place the tube back on the Cell Separation Magnet for 6 to 8 minutes.
-For 17 x 100 mm tube:Place on the Cell Separation Magnet for 8 minutes.
12.Using a new sterile Pasteur pipette, carefully aspirate the supernatant and combine with the enriched fraction from Step 10 above.
13.Repeat Steps 11 and 12. The combined enriched fraction contains T lymphocytes with no bound antibodies or magnetic particles. These cells are ready for downstream applications, or they can be further enriched by proceeding to 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, if desired.
15.To increase the purity of the combined enriched fraction by another 2-4% (compare middle left and middle right panels in the figure), place the tube containing the combined enriched fraction on the Cell Separation Magnet for another 6 to 8 minutes.
-For 17 x 100 mm tube: Place on the Cell Separation Magnet for 8 minutes.
16.Carefully aspirate the supernatant and place in a new sterile tube. This is the twice-enriched fraction. The cells are ready to be processed for downstream applications.
17.Samples of the total cell suspension and the positive and enriched fractions should be analyzed by flow cytometry to evaluate the efficiency of the cell-separation procedure.
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.
- After the final wash, resuspend the cells at a relatively high concentration in 1X BD IMag™ buffer and proceed to step 3.
† Avoid nonspecific labeling by working quickly and adhering to the recommended incubation times.