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Multiparameter flow cytometric analysis of CD8 expression on Human peripheral blood leucocyte populations. Fresh whole blood was treated with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899) to lyse erythrocytes. After washing, the leucocytes were preincubated with BD Pharmingen™ Human BD Fc Block™ (Cat. No. 564219/564220) and then stained with BD Horizon™ BV421 Mouse Anti-Human CD3 antibody (Cat. No. 562426; Top Plots) and with either BD Horizon™ R718 Mouse IgG2a, κ Isotype Control (Cat. No. 566949; Left Plots) or BD Horizon™ R718 Mouse Anti-Human CD8 antibody (Cat. No. 569415/569416; Right Plots). Bivariate pseudocolor density plots showing the correlated expression of CD8 (or Ig Isotype control staining) versus CD3 (Top Plots) or side-light scatter (SSC-A) signals (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of viable leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Multiparameter flow cytometric analysis of CD8 expression on Human peripheral blood leucocyte populations. Fresh whole blood was treated with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899) to lyse erythrocytes. After washing, the leucocytes were preincubated with BD Pharmingen™ Human BD Fc Block™ (Cat. No. 564219/564220) and then stained with BD Horizon™ BV421 Mouse Anti-Human CD3 antibody (Cat. No. 562426; Top Plots) and with either BD Horizon™ R718 Mouse IgG2a, κ Isotype Control (Cat. No. 566949; Left Plots) or BD Horizon™ R718 Mouse Anti-Human CD8 antibody (Cat. No. 569415/569416; Right Plots). Bivariate pseudocolor density plots showing the correlated expression of CD8 (or Ig Isotype control staining) versus CD3 (Top Plots) or side-light scatter (SSC-A) signals (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of viable leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Horizon™ R718 Mouse Anti-Human CD8
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The OKT8 monoclonal antibody specifically recognizes CD8 alpha (CD8a or CD8α). CD8a is a ~32-34 kDa type I transmembrane glycoprotein that is encoded by CD8A, which belongs to the immunoglobulin superfamily (IgSF), and is commonly referred to as CD8. CD8a is expressed by the majority of thymocytes, subpopulations of αβ T cells and γδ T cells, and by subsets of NK cells. Cell surface CD8a is expressed either as a disulfide-linked homodimer (CD8αα) or as a heterodimer (CD8αβ) when disulfide-bonded to a CD8 beta chain (CD8b or 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 coreceptor 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 OKT8 antibody reportedly crossreacts with CD8a expressed by leucocytes from some non-human primate (NHP) species.
Development References (8)
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Hoffman RA, Kung PC, Hansen WP, Goldstein G.. Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood.. Proc Natl Acad Sci U S A. 1980; 77(8):4914-4917. (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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Mattila JT, Diedrich CR, Lin PL, Phuah J, Flynn JL. Simian immunodeficiency virus-induced changes in T cell cytokine responses in cynomolgus macaques with latent Mycobacterium tuberculosis infection are associated with timing of reactivation.. J Immunol. 2011; 186(6):3527-37. (Clone-specific: Flow cytometry). View Reference
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Myburgh JA, Smit JA, Stark JH, Browde S. Total lymphoid irradiation in kidney and liver transplantation in the baboon: prolonged graft survival and alterations in T cell subsets with low cumulative dose regimens.. J Immunol. 1984; 132(2):1019-25. (Clone-specific: Cytotoxicity, Depletion). View Reference
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Perussia B, Fanning V, Trinchieri G. A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8.. J Immunol. 1983; 131(1):223-31. (Clone-specific: Flow cytometry). View Reference
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Reimann KA, Waite BC, Lee-Parritz DE, et al. Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys. Cytometry. 1994; 17(1):102-108. (Clone-specific: Flow cytometry). View Reference
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Reinherz EL. Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, ed. Leukocyte Typing II. New York, NY: Springer-Verlag; 1986:1-549.
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Shiue L, Gorman SD, Parnes JR. A second chain of human CD8 is expressed on peripheral blood lymphocytes.. J Exp Med. 1988; 168(6):1993-2005. (Clone-specific: Flow cytometry). View Reference
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