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Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq. DescriptionPlease see our website for continuous updates regarding the use of this product. The 4B12 monoclonal antibody reacts with mouse CCR7, also known as EBI-1 and CD197. For optimal visualization of CCR7 expression on different cell types it is necessary to use multi-color staining to discriminate different cell subsets (please see Technical Data Sheets for recommended concentration for individual formats). To address specificity, the staining profile of 4B12 has been compared to a polyclonal antibody generated against a CCR7 peptide (Bjorkdahl et al). This analysis confirms that the polyclonal antibody and 4B12 stain similar populations of cells. Furthermore, 4B12 stains mouse CCR7-GFP fusion protein-transfected RBL cells (see data in cat. 14-1971). CCR7 is a chemokine receptor for the chemokines CCL19 (CKß11, ELC, MIP3ß, Scya19, Exodus-3) and CCL21 (CKß9, SLC, MIP2ß, Scya21, Exodus-2). In recent years, the role of chemokines in directing the migration of lymphocytes has been well-characterized. One of the most important mediators of homeostatic trafficking of naïve T cells to secondary lymphoid organs (SLO) is the chemokine receptor CCR7. Binding of its ligands, CCL19 and CCL21, mediates the transendothelial migration of T cells across high endothelial venules into SLO. It has also been demonstrated that CCR7 plays a role in the localization of dendritic cells and B cells during an immune response. In addition to its significant role in the chemotaxis of lymphocytes, human CCR7 has also been recognized as a marker for a distinct subset of memory T cells, the central memory (TCM) population. These cells are characterized by the expression of CCR7 and CD62L and reside within peripheral lymphoid organs. CCR7 also plays a role in thymocyte development and its deficiency leads to disturbed thymic architecture, aberrant T cell development, and limited thymocyte expansion. Applications ReportedFor research use only, not for diagnostic or therapeutic use. This 4B12 antibody has been reported for use in flow cytometric analysis. Applications TestedThe 4B12 antibody has been tested by flow cytometric analysis of C57BL/6, Balb/C and SJL/J splenocytes and thymocytes. Important: Staining with the 4B12 monoclonal antibody requires different conditions than typically used for surface-antigen staining. Please use the protocol below. Moreover, we have found that staining at 37°C, rather than 4°C, results in brighter 4B12 staining, as well as better resolution between positive and negative populations. Please see data for the PE 4B12 (cat. 12-1971) which demonstrates a comparison of staining at 4°C and 37°C. Staining with 4B12 at 37°C is not expected to interfere with co-staining other antigens, however this should be evaluated for individual experiments. 1. Prepare cell suspension as normal and block FcγIIIR/FcγIIR with 5 μg/million cells purified anti-mouse CD16/32 (cat. 14-0161) for 15 minutes on ice. If red blood cell lysis is carried out as part of cell preparation, ensure that fixatives are not present in the red blood cell lysis solution as this will eliminate 4B12 staining. Protocol for RBC Lysis of Mouse Spleen 2. Without washing, add 1 μg/million cells 4B12 and incubate in a 37°C waterbath or at 4°C (please see notes above) for 0.5 hours. 3. Wash cells 1X with 3 mls of Flow Cytometry Staining Buffer (cat. 00-4222) and decant supernatant. 4. Analyze cells on flow cytometer or proceed with secondary staining on ice as normal. Note: Co-staining mouse CCR7 with the 4B12 antibody and the CCR7 ligand CCL19-Fc (cat. 14-1972) may be difficult due to different binding conditions required for the antibody versus the ligand, and steric hindrance which may prevent co-staining of 4B12 and CCL19-Fc. Cross-blocking experiments have demonstrated that 4B12 binding is able to prevent the detectable binding of CCL19-Fc, however not the opposite. Furthermore, the correlation between 4B12 and CCL19-Fc staining may be difficult to predict due to the presence of unknown CCL19-Fc receptors in addition to CCR7. References
Ritter, U, Wiede, F, Mielenz, D, Kiafard, Z, Zwirner, J, and Korner H. 2004. Analysis of the CCR7 expression on murine bone arrow-derived and spleen dendritic cells. Journal of Leukocyte Biology. 76: 472-476. (4B12, FC, Pubmed) Bjorkdahl O, Barber KA, Brett SJ, Daly MG, Plumpton C, Elshourbagy NA, Tite JP, Thomsen LL. 2003. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues. Immunology. 110(2):170-9. Waldner H, Sobel RA, et al. 2006. The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells. J Immunol. 176(9):5455-62. (4B12, FC, PubMed) Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, Blankenstein T, Henning G, Forster R. 2004. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity. 21(2):279-88. (4B12, FC, Pubmed) Related ProductsCat. 14-0161 Affinity Purified anti-mouse CD16/32 - blocks Fc binding (clone 93) Cat. 14-1972 Purified Mouse CCL19-Fc (ELC, MIP3b, CKb11) for mouse CCR7 detection Cat. 11-4317 Streptavidin-FITC (Fluorescein isothiocyanate) Cat. 12-4317 Streptavidin-PE (Phycoerythrin) Cat. 17-4317 Streptavidin Allophycocyanin (SA-APC) Cat. 13-4321 Biotin Rat IgG2a Isotype Control |
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Copyright © 2000-2008 eBioscience, Inc. Product For Research Use Only: Not for further distribution without written consent. |
Mouse Cellular Antigens Chart
