Preparation And Storage
Recommended Assay Procedures
Leukocytes are labeled with BD IMag™ anti-mouse CD90.2 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-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). Place on ice.
Although our experience indicates that the use of Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 (Cat. No. 553141/553142) is not required for optimal cell separation, some laboratories may want to use it in their studies.
If adding Mouse BD Fc Block™, proceed to Step 3. If not adding Mouse BD Fc Block™, proceed to Step 4.
3. Add Mouse BD Fc Block at 0.25 µg/10e6 cells, and incubate on ice for 15 minutes.
4. Wash cells with at least an equal volume of 1X BD IMag™ buffer, and carefully aspirate all the supernatant.
5. Vortex the BD IMag™ anti-mouse CD90.2 Particles - DM thoroughly, and add 50 µl of particles for every 10e7 total cells.
6. MIX THOROUGHLY. Refrigerate at 6°C - 12°C for 30 minutes.
7. Bring the BD IMag-particle labeling volume up to 1 - 8 x 10e7 cells/ml with 1X BD IMag™ buffer, and immediately place the tube on the Cell Separation Magnet. Incubate at room temperature for 6 - 8 minutes.
8. With the tube on the Cell Separation Magnet, carefully aspirate off the supernatant. This supernatant contains the negative fraction.
9. Remove the tube from the Cell Separation Magnet, and add 1X BD IMag™ buffer to the same volume as in Step 7. Gently resuspend cells by pipetting briefly, and return the tube to the Cell Separation Magnet for another 2 - 4 minutes.
10. With the tube on the Cell Separation Magnet, carefully aspirate off the supernatant and discard.
11. Repeat Steps 9 and 10.
12. After the final wash step, resuspend the positive fraction in an appropriate buffer and at an appropriate concentration for further analysis.
NOTE: Avoid nonspecific labeling by working quickly and adhering to recommended incubation times.
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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
BD IMag™ anti-mouse CD90.2 (Thy-1.2) Particles - DM are magnetic nanoparticles that have monoclonal antibody conjugated to their surfaces. These particles are optimized for the positive selection or depletion of CD90.2-bearing leukocytes using the BD IMag™ Cell Separation Magnet. The CD90.2 alloantigen is expressed on thymocytes, most peripheral T lymphocytes, some intraepithelial T lymphocytes (IEL, DEC), epithelial cells, fibroblasts, neurons, hematopoietic stem cells, but not B lymphocytes, of most mouse strains. 30-H12 mAb has been reported not to cross-react with mouse Thy-1.1 (e.g., AKR/J, PL), or with rat Thy-1.
Development References (9)
-
Ikuta K, Uchida N, Friedman J, Weissman IL. Lymphocyte development from stem cells. Annu Rev Immunol. 1992; 10:759-783. (Biology). View Reference
-
LeFrancois L. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire. Immunol Today. 1991; 12(12):436-438. (Biology). View Reference
-
Ledbetter JA, Herzenberg LA. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev. 1979; 47:63-90. (Immunogen). View Reference
-
Ledbetter JA, Rouse RV, Micklem HS, Herzenberg LA. T cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens. Two-parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views. J Exp Med. 1980; 152(2):280-295. (Biology). View Reference
-
Phipps RP, Penney DP, Keng P, et al. Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC. Am J Respir Cell Mol Biol. 1989; 1(1):65-74. (Biology). View Reference
-
Radrizzani M, Carminatti H, Pivetta OH, Idoyaga Vargas VP. Developmental regulation of Thy 1.2 rate of synthesis in the mouse cerebellum. J Neurosci Res. 1995; 42(2):220-227. (Biology). View Reference
-
Tigelaar RE, Lewis JM, Bergstresser PR. TCR gamma/delta+ dendritic epidermal T cells as constituents of skin-associated lymphoid tissue. J Invest Dermatol. 1990; 94(6):58S-63S. (Biology). View Reference
-
Zheng B, Han S, Kelsoe G. T helper cells in murine germinal centers are antigen-specific emigrants that downregulate Thy-1. J Exp Med. 1996; 184(3):1083-1091. (Biology). View Reference
-
Zhong RK, Donnenberg AD, Edison L, Harrison DE. The appearance of Thy-1- donor T cells in the peripheral circulation 3-6 weeks after bone marrow transplantation suggests an extrathymic origin. Int Immunol. 1996; 8(2):171-176. (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.
