Description
The 4G7 monoclonal antibody specifically binds to the ~90-95 kDa type I transmembrane CD19 glycoprotein which is also known as B-lymphocyte surface antigen B4 (B4) or Leu-12. CD19 is expressed during all stages of B-cell maturation and differentiation, except on plasma cells. CD19 is also present on follicular dendritic cells. It is not found on T cells or on normal granulocytes. CD19 is a signal transduction molecule that regulates B cell development, activation, proliferation and differentiation. It associates with the complement receptor 2 (CD21), TAPA-1 (CD81), Leu 13, and/or MHC class II to form a signal transduction complex on the surface of B cells.
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 (Cat. No. 552311).
Format
- FormatBD IMag™-DM
Suggested Companion Products
Cell Separation Magnet RUO Cat No: 552311
Buffer (10X) RUO 100mLCat No: 552362
APC Mouse Anti-Human CD19 HIB19RUO 100TestsCat No: 555415
FITC Mouse Anti-Human CD3 UCHT1 (also known as UCHT-1; UCHT 1)RUO 100TestsCat No: 555916
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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
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- 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.
Peripheral blood mononuclear cells (PBMC) are labeled with BD IMag™ anti-human CD19 Particles - DM according to the following protocol. This labeled cell suspension is then placed within the magnetic field of the 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, the small size of the magnetic particles allows the positive fraction to be further evaluated in downstream applications such as flow cytometry system.
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-ìm 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 10e7 total cells.
5. MIX THOROUGHLY. Incubate at room temperature for 30 minutes.
6. Bring the BD IMag™-particle labeling volume up to a concentration of 1 - 8 x 107 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 1 ml of 1X BD IMag™ buffer. 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).
NOTES:
*A more detailed protocol for the magnetic cell separation appears on the Technical Data Sheet accompanying the Cell Separation Magnet. The concentration of BD IMag™ anti-human CD19 Particles - DM suggested in the 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.
*Avoid nonspecific labeling by working quickly and keeping incubation times to a minimum.
