Description
BD IMag™ anti-mouse CD117 Magnetic Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces.These particles are optimized for the positive selection or depletion of CD117-bearing cells using the BD IMag™ Cell Separation Magnet.The 2B8 antibody reacts with CD117 (c-Kit), a transmembrane tyrosine-kinase receptor which is encoded by the Kit gene (formerly dominant white spotting, W). The c-Kit ligand (also known as steel factor, stem cell factor, and mast cell growth factor) encoded by the Kit1 gene (formerly steel, SI), is a co-mitogen for hematopoietic stem cells, myeloerythroid progenitors and a mast-cell differentiation factor. The KitW and Kit1SI mutant alleles have similar pleiotropic effects on the development of melanocytes, germ cells, and the hematopoietic system. In the adult bone marrow, CD117 is expressed on hematopoietic progenitor cells, including CD90 (Thy-1) low, TER-119-, CD45R/B220-, CD11b (Mac-1)-, Ly-6G (Gr-1)-, CD4-, CD8-, and Sca-1 (Ly-6A/E)+ multipotent hemotopoietic stem cells, progenitors committed to myeliod and/or erythroid lineages, and precursors of B and T lymphocytes. This widespread expression of CD117 in hematopoietic precursors is consistent with the participation of c-Kit and its ligand in the regulation of several hematopoietic lineages. Intrathymic expression of c-Kit and c-Kit ligand suggest that CD117 is also involved in the regulation of some events during the development of T lymphocytes. CD117 is also expressed by mast cells and by dendritic cells found in the periarteriolar lymphocytoc sheaths (T-cell areas) of splenic white pulp. The mAb 2B8 reportedly does not block the action of c-Kit.
Format
- FormatBD IMag™-DM
Suggested Companion Products
APC Rat Anti-Mouse CD45 30-F11RUO 0.1mgCat No: 559864
PE Rat Anti-Mouse CD117 ACK45RUO 0.2mgCat No: 553869
Cell Separation Magnet RUO Cat No: 552311
Buffer (10X) RUO 100mLCat No: 552362
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light.Do not freeze.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
- 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.
- 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/pharmingen/protocols for technical protocols.
A single-cell suspension from the lymphoid tissue of interest is labeled with BD IMag™ anti-mouse CD117 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 a single-cell suspension from the lymphoid tissue of interest according to standard laboratory procedures. Remove clumps of cells and/or debris by passing the suspension through a 70-mm 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-mouse CD117 Magnetic Particles - DM thoroughly, and add 50 μl of particles for every 10 million total cells.
5. MIX THOROUGHLY.Refrigerate at 6°C to 12°C for 30 minutes.
6. Bring the BD IMag™-particle labeling volume up to 10 to 80 million cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the Cell Separation Magnet.Incubate at room temperature for 8-10 minutes.
7.With the tube on the Cell Separation Magnet, carefully pipette 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 pipette 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).
NOTE:The concentration of BD IMag™ anti-mouse CD117 Particles - DM suggested in the protocol has been optimized for the purification of CD117-positive cells from mouse bone marrow.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-mouse CD117 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 as recommended.
