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Multiparameter flow cytometric analysis of CD4 expression on human peripheral blood leucocyte populations. Whole blood was stained with either BD Horizon™ BV421 Mouse IgG2a, k Isotype Control (Cat. No. 562439; Left Plot) or BD Horizon BV421 Mouse Anti-Human CD3 antibody (Cat. No. 566696/566697; Right Plot). Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). A two-parameter pseudocolor density plot showing the correlated expression of CD3 (or Ig Isotype control staining) versus side-light scatter (SSC-A) signals was derived from gated events with the forward and side-light scatter characteristics of viable leucocyte populations. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.
BD Horizon™ BV421 Mouse Anti-Human CD3
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.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime BD Horizon Brilliant dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- BD Horizon Brilliant Violet 421 is covered by one or more of the following US patents: 8,158,444; 8,362,193; 8,575,303; 8,354,239.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
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- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
Companion Products
The OKT3 monoclonal antibody specifically recognizes the CD3 epsilon subunit (CD3e/CD3ε) of the CD3 complex which consists of four transmembrane proteins (γ, δ, ε, ζ) that are associated with the T cell antigen receptor (TCR) to form the CD3/TCR complex. The CD3 complex associates with either TCR αβ or TCR γδ heterodimers that are alternatively expressed by some thymocytes, T cells or NKT cells. The CD3 complex is required for the cell surface expression and signal-transducing functions of the TCR. The CD3 complex is expressed by ~60-85% thymocytes and by all peripheral mature T cells. CD3e is also known as T3E or TCRE. CD3e is a ~20 kDa unglycosylated type I transmembrane protein that is encoded by CD3E which belongs to the immunoglobulin superfamily (IgSF). CD3e has an Ig-like extracellular domain (ECD) and an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The OKT3 antibody can reportedly fix complement, stimulate T cell proliferation and cytokine production, and block the binding of other human CD3e-specific antibodies including UCHT1 and SK7.
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument's configurations (lasers and optical filters) are appropriate for this dye.
Development References (12)
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Burns GF, Boyd AW, Beverley PC. Two monoclonal anti-human T lymphocyte antibodies have similar biologic effects and recognize the same cell surface antigen. J Immunol. 1982; 129(4):1451-1457. (Clone-specific: Blocking, Functional assay, Immunoprecipitation, Radioimmunoassay). View Reference
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Emmrich F. Selective stimulation of human CD4 and CD8 T-cells by crosslining the T-cell receptor with subset-specific differentiation antigens. In: McMichael AJ. A.J. McMichael .. et al., ed. Leucocyte typing III : white cell differentiation antigens. Oxford New York: Oxford University Press; 1987:203-206.
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Ernst DN, Shih CC. CD3 complex. J Biol Regul Homeost Agents. 2000; 14(3):226-229. (Biology). View Reference
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Horibe K, Knowles RW, Naito K, Morishima Y, Dupont B. Analysis of T lymphocyte antibody specificities: Comparison of serology with immunoprecipitation patterns. In: Bernard A. A. Bernard .. et al., ed. Leucocyte typing : human leucocyte differentiation antigens detected by monoclonal antibodies. Berlin New York: Springer-Verlag; 1984:212-224.
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Kung P, Goldstein G, Reinherz EL, Schlossman SF. Monoclonal antibodies defining distinctive human T cell surface antigens. Science. 1979; 206(4416):347-349. (Immunogen: Cytotoxicity, Flow cytometry, Radioimmunoassay). View Reference
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Kurrle R, Seyfert W, Trautwein A, Seiler FR. T cell activation by CD3 antibodies. In: Reinherz EL. Ellis L. Reinherz .. et al., ed. Leukocyte typing II. New York: Springer-Verlag; 1986:137-146.
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Li B, Wang H, Dai J, et al. Construction and characterization of a humanized anti-human CD3 monoclonal antibody 12F6 with effective immunoregulation functions. Immunology. 2005; 116(4):487-498. (Clone-specific: Blocking, Flow cytometry). View Reference
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Semnani R, Nutman TB, Corrado G, Hochman P, Shaw S, Van Seventer GA. Costimulation mediated by purified ICAM-1 and LFA-3 regulates differential stimulation and cytokine secretion of human 'naive' and 'memory' CD4+ T cells. In: Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens. Oxford: Oxford University Press; 1995:1488-1491.
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Touraine JL, Favrot MC, Ansary ME, Cordier G, de bouteiller O. Phenotype of prothymocytes from human bone marrow determined by monoclonal antibodies: Modification induced by thymic factots. In: Bernard A. A. Bernard .. et al., ed. Leucocyte typing : human leucocyte differentiation antigens detected by monoclonal antibodies. Berlin New York: Springer-Verlag; 1984:298-311.
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Tunnacliffe A, Olsson C, Traunecker A, Krissansen GW, Karjalainen K, de la Hera A. The majority of CD3 epitopes are conferred by the epsilon chain. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:295-296.
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Van Wauwe JP, De Mey JR, Goossens JG. OKT3: a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J Immunol. 1980; 124:2708-2713. (Clone-specific: Functional assay). View Reference
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Van Wauwe JP, Goossens JG, Beverley PC. Human T lymphocyte activation by monoclonal antibodies; OKT3, but not UCHT1, triggers mitogenesis via an interleukin 2-dependent mechanism. J Immunol. 1984; 133(1):129-132. (Clone-specific: Functional assay). View Reference
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