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Identification of CD34+ and CD34- subpopulations of hematopoietic progenitors. BALB/c bone marrow cells were treated with Mouse BD Fc Block™ purified anti-CD16/CD32 mAb 2.4G2 (Cat. No. 553141/553142) and stained with either Alexa Fluor® 647-conjugated anti-mouse CD34 (Clone RAM34, Cat. No. 560230/560233, right panel) or a Alexa Fluor® 647-conjugated rat IgG2a, κ isotype control (clone R35-95, Cat. No. 557690, left panel), followed by staining with a PE Lineage Cocktail consisting of CD3e, CD11b, CD45R/B220, Gr-1 [Ly6G, Ly6C] mAbs (Cat. No. 553063/553064/561824, 553311/557397/561689, 553089/553090/561878, 553128/561084, respectively) to identify major lineage committed cells. The contour plots were derived from the gated events based on light scattering characteristics of leukocytes and fluorescence characteristics of the CD34 and Lineage markers . Flow cytometry was performed on a BD FACSCanto™ System.
BD Pharmingen™ Alexa Fluor® 647 Rat anti-Mouse CD34
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Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
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- Alexa Fluor™ is a trademark of Life Technologies Corporation.
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The RAM34 monoclonal antibody specifically binds to the CD34 glycoprotein on the surface of three independently derived mouse CD34-transfected cell lines. RAM34 antibody also reacts with the mouse cell lines PA6, 416B, Swiss 3T6, NIH, 3T3, DA1, and M1, all of which are positive for expression of mouse CD34 mRNA. Cell lines shown to be negative for CD34 mRNA transcript, including WEHI-3B, EL4, 18.8, and CMT64/61, are also negative for surface expression of CD34 as determined by RAM34 staining. Normal thymocytes and splenocytes are negative for CD34 expression. In the bone marrow, 7-10% of cells are stained with the RAM34 antibody, including most of the Ly-6A/E (Sca-1)+ CD90 (Thy-1)low Lineage Marker- hematopoietic stem cell-enriched subpopulation and myeloerythroid progenitors. CD34 is also expressed on a small percentage of fetal liver cells, including NK-cell progenitors. CD34 has been reported to be expressed on the endothelium of capillaries and, in this form, to function as a ligand for L-selectin. Consistent with this observation, RAM34 antibody stains endothelial cells in spleen, thymus, and postcapillary HEVs in the lymph nodes. It is reported that RAM34 antibody can be used to select CD34+ CD117 (c-Kit)+ Ly-6A/E (Sca-1)+ Lineage Marker- bone marrow-derived hematopoietic stem cells, capable of short-term multi-lineage reconstitution of lethally irradiated mice. In contrast, the CD34- CD117+ Sca-1+ Lineage Marker- population contains self-renewing hematopoietic stem cells. Similarly, the bone marrow population with high dye-efflux capacity and highly enriched for long-term reconstituting hematopoietic stem cells, is CD34- CD117 (c-Kit)+ Ly-6A/E (Sca-1)+ Lineage Marker-.
Development References (11)
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Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000; 404(6774):193-197. (Clone-specific: Flow cytometry). View Reference
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Baumheter S, Singer MS, Henzel W, et al. Binding of L-selectin to the vascular sialomucin CD34. Science. 1993; 262(5132):436-438. (Biology). View Reference
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Brown J, Greaves MF, Molgaard HV. The gene encoding the stem cell antigen, CD34, is conserved in mouse and expressed in haemopoietic progenitor cell lines, brain, and embryonic fibroblasts. Int Immunol. 1991; 3(2):175-184. (Biology). View Reference
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Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med. 1997; 3(12):1337-1345. (Clone-specific: Flow cytometry). View Reference
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Lorenz K, Grashoff C, Torka R, et al. Integrin-linked kinase is required for epidermal and hair follicle morphogenesis. J Cell Biol. 2007; 177(3):501-513. (Biology: Immunofluorescence, Immunohistochemistry). View Reference
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Lu J, Patrene KD, Herberman RB, Boggs SS. Expression of murine CD34 by fetal liver NK cell progenitors. Exp Hematol. 1999; 27(2):272-281. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Morel F, Szilvassy SJ, Travis M, Chen B, Galy A. Primitive hematopoietic cells in murine bone marrow express the CD34 antigen. Blood. 1996; 88(10):3774-3784. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 1996; 273(5272):242-245. (Immunogen: Flow cytometry). View Reference
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Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science. 1988; 241(4861):58-62. (Biology). View Reference
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Suda J, Sudo T, Ito M, Ohno N, Yamaguchi Y, Suda T. Two types of murine CD34 mRNA generated by alternative splicing. Blood. 1992; 79(9):2288-2295. (Biology). View Reference
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Suzuki A, Andrew DP, Gonzalo JA, et al. CD34-deficient mice have reduced eosinophil accumulation after allergen exposure and show a novel crossreactive 90-kD protein. Blood. 1996; 87(9):3550-3562. (Clone-specific: Immunoaffinity chromatography, Immunohistochemistry, Immunoprecipitation). View Reference
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