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BD Horizon™ BB515 Mouse Anti-Human CD8
Clone RPA-T8 (RUO)
Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. Human whole blood was stained with either BD Horizon™ BB515 Mouse IgG1, κ Isotype Control (Cat. No. 564416; dashed line histogram) or BD Horizon BB515 Mouse Anti-Human CD8 antibody (Cat. No. 564526/564527; bold solid line histogram). Alternatively, cells were stained with FITC Anti-Human CD8 antibody (Cat. No. 555366/557085/561947/561948; thin solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202).
Overlaid histograms are shown to facilitate staining comparisons between: BB515 Anti-CD8 antibody versus its Ig Isotype Control (Left Panel), and BB515 Anti-CD8 antibody versus FITC Anti-CD8 antibody (Right Panel). The fluorescence histograms showing CD8 expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. Human whole blood was stained with either BD Horizon™ BB515 Mouse IgG1, κ Isotype Control (Cat. No. 564416; dashed line histogram) or BD Horizon BB515 Mouse Anti-Human CD8 antibody (Cat. No. 564526/564527; bold solid line histogram). Alternatively, cells were stained with FITC Anti-Human CD8 antibody (Cat. No. 555366/557085/561947/561948; thin solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202).
Overlaid histograms are shown to facilitate staining comparisons between: BB515 Anti-CD8 antibody versus its Ig Isotype Control (Left Panel), and BB515 Anti-CD8 antibody versus FITC Anti-CD8 antibody (Right Panel). The fluorescence histograms showing CD8 expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. Human whole blood was stained with either BD Horizon™ BB515 Mouse IgG1, κ Isotype Control (Cat. No. 564416; dashed line histogram) or BD Horizon BB515 Mouse Anti-Human CD8 antibody (Cat. No. 564526/564527; bold solid line histogram). Alternatively, cells were stained with FITC Anti-Human CD8 antibody (Cat. No. 555366/557085/561947/561948; thin solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202).
Overlaid histograms are shown to facilitate staining comparisons between: BB515 Anti-CD8 antibody versus its Ig Isotype Control (Left Panel), and BB515 Anti-CD8 antibody versus FITC Anti-CD8 antibody (Right Panel). The fluorescence histograms showing CD8 expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. Human whole blood was stained with either BD Horizon™ BB515 Mouse IgG1, κ Isotype Control (Cat. No. 564416; dashed line histogram) or BD Horizon BB515 Mouse Anti-Human CD8 antibody (Cat. No. 564526/564527; bold solid line histogram). Alternatively, cells were stained with FITC Anti-Human CD8 antibody (Cat. No. 555366/557085/561947/561948; thin solid line histogram). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202).
Overlaid histograms are shown to facilitate staining comparisons between: BB515 Anti-CD8 antibody versus its Ig Isotype Control (Left Panel), and BB515 Anti-CD8 antibody versus FITC Anti-CD8 antibody (Right Panel). The fluorescence histograms showing CD8 expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Horizon™
CD8α; CD8A; CD8 alpha; Leu2; MAL; T8; p32
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse IgG1, κ
Human CD8a
Flow cytometry (Routinely Tested)
5 µl
IV T171; V T-CD08.03; VI 6T-CD8.1, 6T-081
AB_2744458
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with BD Horizon™ BB515 under optimum conditions and unconjugated antibody was removed.
Recommended Assay Procedures
BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to 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 CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.
For optimal results, it is recommended to perform 2 washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.
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).
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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Brilliant Stain Buffer RUO
Size
1000 Tests
Cat No.
566349
Brilliant Stain Buffer Plus RUO
Size
1000 Tests
Cat No.
566385
Stain Buffer (FBS) RUO
Size
500 ML
Cat No.
554656
Brilliant Stain Buffer RUO
Size
100 Tests
Cat No.
563794
Lysing Solution 10X Concentrate IVD
Size
100 ML
Cat No.
349202
Stain Buffer (BSA) RUO
Size
500 ML
Cat No.
554657
564527 Rev. 5
Antibody Details
RPA-T8
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 BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor® 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.
564527 Rev. 5
Format Details
BB515
The BD Horizon Brilliant™ Blue 515 (BB515) dye is part of the BD Horizon Brilliant™ Blue family of dyes. This dye is a polymer fluorochrome with an excitation maximum (Ex Max) at 490-nm and an emission maximum (Em Max) of 515-nm. Driven by BD innovation, BB515 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 520-nm (e.g., 530/30-nm). BB515 reagents are significantly brighter than equivalent FITC or Alexa Fluor™ 488 reagents with less spillover into the PE detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye. It is not recommended to use BB515 in cocktails that include streptavidin conjugates as it may cause high background.
BB515
490 nm
515 nm
564527 Rev.5
Citations & References
View product citations for antibody "564527" on CiteAb
Development References (3)
-
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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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.
-
Zola H. Leukocyte and stromal cell molecules : the CD markers. Hoboken, N.J.: Wiley-Liss; 2007.
564527 Rev. 5
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.