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Anti-Mouse CD90.2 Magnetic Particles - DM
Anti-Mouse CD90.2 Magnetic Particles - DM

Positive selection and depletion of mouse CD90.2-positive splenocytes. Leukocytes were labeled with BD IMag™ anti-mouse CD90.2 Particles - DM (Cat. No. 551518) as described in the protocol. After labeling, the cells were separated using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), and the negative (CD90.2-) and positive (CD90.2+) fractions were collected. Please refer to the Separation Flow Chart to identify the separated cell populations represented in this figure. For flow cytometric analysis, fresh splenocytes (left panel), the negative fraction (middle panel), and the positive fraction (right panel) were stained with FITC Anti-Mouse CD3e (Cat. No. 553061) and PE Anti-mouse CD90.2 (Cat. No. 553005). The percent CD3e+/CD90.2+ cells in each sample is given in the upper right corner.

Positive selection and depletion of mouse CD90.2-positive splenocytes. Leukocytes were labeled with BD IMag™ anti-mouse CD90.2 Particles - DM (Cat. No. 551518) as described in the protocol. After labeling, the cells were separated using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), and the negative (CD90.2-) and positive (CD90.2+) fractions were collected. Please refer to the Separation Flow Chart to identify the separated cell populations represented in this figure. For flow cytometric analysis, fresh splenocytes (left panel), the negative fraction (middle panel), and the positive fraction (right panel) were stained with FITC Anti-Mouse CD3e (Cat. No. 553061) and PE Anti-mouse CD90.2 (Cat. No. 553005). The percent CD3e+/CD90.2+ cells in each sample is given in the upper right corner.

Product Details
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BD IMag™
Thy-1.2; T25; Thymus Cell Antigen 1; Theta
Mouse (QC Testing)
Rat LOU, also known as Louvain, LOU/C, LOU/M IgG2b, κ
Mouse Thymus / Spleen
Cell separation (Routinely Tested)
21838
AB_398514
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


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.

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

  1. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  2. 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.
  3. 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.
  4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
551518 Rev. 6
Antibody Details
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30-H12

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.

551518 Rev. 6
Format Details
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BD IMag DM
BD IMag DM
551518 Rev.6
Citations & References
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Development References (9)

  1. Ikuta K, Uchida N, Friedman J, Weissman IL. Lymphocyte development from stem cells. Annu Rev Immunol. 1992; 10:759-783. (Biology). View Reference
  2. 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
  3. Ledbetter JA, Herzenberg LA. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev. 1979; 47:63-90. (Immunogen). View Reference
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
View All (9) View Less
551518 Rev. 6

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