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Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes. Human whole blood was stained with either BD Horizon™ BUV563 Mouse IgG1, κ Isotype Control (Cat. No. 612920; dashed line histogram) or BD Horizon™ BUV563 Mouse Anti-Human CD8 antibody (Cat. No. 612914/612915; solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The fluorescence histogram showing CD8 expression (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Horizon™ BUV563 Mouse Anti-Human CD8
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 CompBead 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 two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. 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).
Note: When using high concentrations of antibody, background binding of this dye to erythroid cell subsets (mature erythrocytes and precursors) has been observed. For researchers studying these cell populations, or in cases where light scatter gating does not adequately exclude these cells from the analysis, this background may be an important factor to consider when selecting reagents for panel(s).
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- 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.
- BD Horizon Brilliant Ultraviolet 563 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,575,303; 8,354,239.
- CF™ is a trademark of Biotium, Inc.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The RPA-T8 monoclonal antibody specifically binds to CD8 alpha (CD8α). CD8α is a type I transmembrane glycoprotein and a member of the immunoglobulin superfamily. CD8α is expressed by the majority of thymocytes, by subpopulations of αβ T cells and γδ T cells and by some NK cells. Cell surface CD8α is expressed either as a disulfide-linked homodimer (CD8αα) or as a heterodimer (CD8αβ) when disulfide-bonded to a CD8 beta chain (CD8β). CD8-positive αβ T cells coexpress both CD8αα homodimers and CD8αβ heterodimers whereas some γδ T cells and NK cells express CD8αα homodimers. CD8 plays important roles in T cell activation and selection. The extracellular IgSF domain of CD8α binds to a non-polymorphic determinant on HLA class I molecules (α3 domain) and enables CD8 to function as a co-receptor with MHC class I-restricted TCR during T cell recognition of antigen. The cytoplasmic domain of CD8α associates with Lck, a Src family protein tyrosine kinase that is involved in intracellular signaling. The RPA-T8 and HIT8a monoclonal antibodies are not cross-blocking. This clone has been reported to react with a subset of peripheral blood lymphocytes, but not monocytes nor granuloyctes, of baboon and both rhesus and cynomolgus macaque monkey. In general, a higher frequency of CD8+ and CD4+CD8+ lymphocytes are observed in non-human primates compared to normal human donors.
The antibody was conjugated to BD Horizon BUV563 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 which has an Ex Max of 348 nm and an acceptor dye. The tandem has an Em Max at 563 nm. BD Horizon BUV563 can be excited by the 355 nm ultraviolet laser. On instruments with a 561 nm Yellow-Green laser, the recommended bandpass filter is 585/15 nm with a 535 nm long pass to minimize laser light leakage. When BD Horizon BUV563 is used with an instrument that does not have a 561 nm laser, a 560/40 nm filter with a 535 nm long pass may be more optimal. Due to the excitation and emission characteristics of the acceptor dye, there may be spillover into the PE and PE-CF594 detectors. However, the spillover can be corrected through compensation as with any other dye combination.
Development References (8)
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Garbrecht F, Loebel A, Disanto JP, Flomenberg N. Chatacterization of Workshop antiCD8 mAb using human CD8-expressing murine L-cell transfectants. In: Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995:354-356.
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Herodin F, Thullier P, Garin D, Drouet M. Nonhuman primates are relevant models for research in hematology, immunology and virology. Eur Cytokine Netw. 2005; 16(2):104-116. (Clone-specific: Flow cytometry). View Reference
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Kersh EN, Kersh GJ, Allen PM. Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation. J Exp Med. 1999; 190(11):1627-1636. (Clone-specific: Flow cytometry). View Reference
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Kishimoto T. Tadamitsu Kishimoto .. et al., ed. Leucocyte typing VI : white cell differentiation antigens : proceedings of the sixth international workshop and conference held in Kobe, Japan, 10-14 November 1996. New York: Garland Pub.; 1997.
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Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
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Lin G-X, Yang X, Hollemweguer E, et al. Cross-reactivity of CD antibodies in eight animal species. In: Mason D. David Mason .. et al., ed. Leucocyte typing VII : white cell differentiation antigens : proceedings of the Seventh International Workshop and Conference held in Harrogate, United Kingdom. Oxford: Oxford University Press; 2002:519-523.
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Rabin RL, Park MK, Liao F, Swofford R, Stephany D, Farber JM. Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling. J Immunol. 1999; 162(7):3840-3850. (Clone-specific: Flow cytometry). View Reference
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Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995.
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.