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 is conjugated to BD Horizon V500, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser with an Ex max of 415 nm and Em Max at 500 nm. BD Horizon V500 conjugates emit at a similar wavelength to Amcyan yet exhibit reduced spillover into the FITC channel. For more information on BD Horizon V500, visit bdbiosciences.com/colors.
When compensating dyes in this spectral range (such as BD Horizon V500 and AmCyan), the most accurate compensation can be obtained using single stained cellular controls. Due to spectral differences between cells and beads in this channel, using BD™ CompBeads can result in spillover errors for V500 and AmCyan reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different V500 reagents (eg, CD4 vs. CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone specific compensation controls when using these reagents.