Preparation And Storage
Recommended Assay Procedures
Peripheral Blood Mononuclear Cells (PBMC) are labeled with BD IMag™ anti-human CD19 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 PBMC from anti-coagulated human blood, preferably by density gradient centrifugation using Ficoll-Paque™. 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, 2 mM EDTA, and 0.09% sodium azide). Store at 4°C.
3. Wash cells with an excess volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.
4. Vortex the BD IMag™ anti-human CD19 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 media, and proceed with desired downstream application(s).
The concentration of BD IMag™ anti-human CD19 Particles - DM suggested in this protocol has been optimized for the purification of CD19 positive B lymphocytes from human peripheral blood. When labeling target cell populations present at lower frequencies, fewer BD IMag™ particles can be used. Conversely, when labeling target cell populations that are present at higher frequencies, more particles should be used. To determine the optimal concentration of the BD IMag™ anti-human CD19 Particles - DM for a particular application, a titration in two-fold increments is recommended.
NOTE: Avoid nonspecific labeling by working quickly and keeping incubation times to a minimum.
Product Notices
- 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.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
BD IMag™ anti-human CD19 Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. These particles are optimized for the positive selection or depletion of CD19-bearing leukocytes using the BD IMag™ Cell Separation Magnet. CD19 is expressed during all stages of B-cell differentiation and maturation, except plasma cells. CD19 is also present on follicular dendritic cells. It is not found on T cells or on normal granulocytes.
Development References (6)
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Bradbury LE, Goldmacher VS, Tedder TF. The CD19 signal transduction complex of B lymphocytes. Deletion of the CD19 cytoplasmic domain alters signal transduction but not complex formation with TAPA-1 and Leu 13. J Immunol. 1993; 151(6):2915-2927. (Biology). View Reference
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Favaloro EJ, Moraitis N, Koutts J, Exner T, Bradstock KF. Endothelial cells and normal circulating haemopoietic cells share a number of surface antigens. Thromb Haemost. 1989; 61(2):217-224. (Biology). View Reference
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Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
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Nadler LM, Anderson KC, Marti G, et al. B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes. J Immunol. 1983; 131(1):244-250. (Biology). View Reference
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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.
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Uckun FM, Muraguchi A, Ledbetter JA, et al. Biphenotypic leukemic lymphocyte precursors in CD2+CD19+ acute lymphoblastic leukemia and their putative normal counterparts in human fetal hematopoietic tissues. Blood. 1989; 73(4):1000-1015. (Biology). View Reference
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.
