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Flow cytometric analysis using BD OptiBuild™ RB780 Hamster Anti-Mouse CD69 antibody (Cat. No. 755598; solid line histogram) on viable C57BL/6 Mouse splenocytes stimulated with PMA for 5 hours, with Autofluorescence Control (dotted line histogram). Flow cytometry was performed using a BD LSRFortessa™ X-20 Flow Cytometer System.
BD OptiBuild™ RB780 Hamster Anti-Mouse CD69
Regulatory Status Legend
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
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Researchers should determine the optimal concentration of this reagent for their individual applications.
- The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- 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 http://regdocs.bd.com to access safety data sheets (SDS).
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Companion Products
The H1.2F3 monoclonal antibody specifically binds to CD69 (Very Early Activation antigen), an 85 kDa disulfide-linked homodimer of differentially glycosylated subunits. CD69 is a C-type lectin, most closely related to the NKR-P1 and Ly-49 NK cell-activation molecules. Its expression is rapidly induced upon activation of lymphocytes (T, B, NK, and NK-T cells), neutrophils, and macrophages. CD69 is expressed also on thymocytes that are undergoing positive selection; its role in that process is unclear. H1.2F3 mAb augments PMA-induced T-cell stimulation and IFN-γ-induced macrophage stimulation. IL-2-activated NK cells express CD69, and H1.2F3 mAb induces redirected lysis of FcR-bearing target cells by NK cells.
Development References (15)
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Bendelac A, Matzinger P, Seder RA, Paul WE, Schwartz RH. Activation events during thymic selection. J Exp Med. 1992; 175(3):731-742. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Gabor MJ, Godfrey DI, Scollay R. Recent thymic emigrants are distinct from most medullary thymocytes. Eur J Immunol. 1997; 27(8):2010-2050. (Clone-specific: Flow cytometry). View Reference
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Karlhofer FM, Yokoyama WM. Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways. J Immunol. 1991; 146(10):3662-3673. (Clone-specific: Induction). View Reference
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Keefe R, Dave V, Allman D, Wiest D, Kappes DJ. Regulation of lineage commitment distinct from positive selection. Science. 1999; 286(5442):1149-1153. (Biology). View Reference
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Lauzurica P, Sancho D, Torres M, et al. Phenotypic and functional characteristics of hematopoietic cell lineages in CD69-deficient mice. Blood. 2000; 95(7):2312-2320. (Clone-specific: Flow cytometry). View Reference
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Marzio R, Jirillo E, Ransijn A, Mauel J, Corradin SB. Expression and function of the early activation antigen CD69 in murine macrophages. J Leukoc Biol. 1997; 62(3):349-355. (Clone-specific: Activation). View Reference
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Merkenschlager M, Graf D, Lovatt M, Bommhardt U, Zamoyska R, Fisher AG. How many thymocytes audition for selection. J Exp Med. 1997; 186(7):1149-1158. (Clone-specific: Flow cytometry). View Reference
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Nishimura T, Kitamura H, Iwakabe K, et al. The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes. Int Immunol. 2000; 12(7):987-994. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Punt JA, Suzuki H, Granger LG, Sharrow SO, Singer A. Lineage commitment in the thymus: only the most differentiated (TCRhibcl-2hi) subset of CD4+CD8+ thymocytes has selectively terminated CD4 or CD8 synthesis. J Exp Med. 1996; 184(6):2091-2099. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Sobel ES, Yokoyama WM, Shevach EM, Eisenberg RA, Cohen PL. Aberrant expression of the very early activation antigen on MRL/Mp-lpr/lpr lymphocytes. J Immunol. 1993; 150(2):673-682. (Clone-specific: Stimulation). View Reference
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Wilkinson RW, Anderson G, Owen JJ, Jenkinson EJ. Positive selection of thymocytes involves sustained interactions with the thymic microenvironment. J Immunol. 1995; 155(11):5234-5240. (Clone-specific: Cell separation, Flow cytometry). View Reference
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Yokoyama WM, Koning F, Kehn PJ, et al. Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation. J Immunol. 1988; 141(2):369-376. (Immunogen: Flow cytometry, Immunoprecipitation, Stimulation). View Reference
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Yokoyama WM, Maxfield SR, Shevach EM. Very early (VEA) and very late (VLA) activation antigens have distinct functions in T lymphocyte activation. Immunol Rev. 1989; 109:153-176. (Clone-specific: Flow cytometry, Stimulation). View Reference
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Ziegler SF, Levin SD, Johnson L, et al. The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex. J Immunol. 1994; 152(3):1228-1236. (Clone-specific: Flow cytometry). View Reference
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Ziegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells. 1994; 12(5):456-465. (Biology: Flow cytometry). 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.