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PE Rat Anti-Mouse CD8a
PE Rat Anti-Mouse CD8a

Two-color flow cytometric analysis of CD8a expression on mouse splenocytes. BALB/c mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142) and then stained with APC Armenian Hamster Anti-Mouse CD3e (Cat. No. 553066/561826) and with either PE Rat IgG2b, κ Isotype Control (Cat. No. 555848; Left Plot) or PE Rat Anti-Mouse CD8a (Cat. No. 567630; Right Plot) at 0.5 μg/test. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD8a (or Ig Isotype control staining) versus CD3e was derived from gated events with the forward and side light-scatter characteristics of viable (DAPI-negative) leucocytes. Flow cytometry and data analysis were performed using a BD FACSCelesta™ Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Two-color flow cytometric analysis of CD8a expression on mouse splenocytes. BALB/c mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142) and then stained with APC Armenian Hamster Anti-Mouse CD3e (Cat. No. 553066/561826) and with either PE Rat IgG2b, κ Isotype Control (Cat. No. 555848; Left Plot) or PE Rat Anti-Mouse CD8a (Cat. No. 567630; Right Plot) at 0.5 μg/test. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD8a (or Ig Isotype control staining) versus CD3e was derived from gated events with the forward and side light-scatter characteristics of viable (DAPI-negative) leucocytes. Flow cytometry and data analysis were performed using a BD FACSCelesta™ Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Product Details
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BD Pharmingen™
Cd8a; CD8 antigen, alpha chain; CD8a; CD8α; Ly-2; Lyt2; Lyt-2
Mouse (QC Testing)
Rat SD, also known as Sprague-Dawley (outbred) IgG2b, λ
Concanavalin A-stimulated BALB/c Splenic T Cell Blasts
Flow cytometry (Routinely Tested)
0.2 mg/ml
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 to the dye under optimum conditions and unconjugated antibody and free dye were removed.

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.

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  6. An isotype control should be used at the same concentration as the antibody of interest.
567630 Rev. 1
Antibody Details
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5H10-1

The 5H10-1 monoclonal antibody specifically recognizes CD8a which is also known as  CD8 alpha (CD8α), Ly-2 or Lyt-2. CD8a is an ~34 kDa single pass type I transmembrane glycoprotein that is encoded by Cd8a (CD8 antigen, alpha chain) which belongs to the immunoglobulin superfamily (IgSF). CD8a is comprised of an extracellular region that contains an IgV-like domain followed by a transmembrane region and cytoplasmic tail with a Lck tyrosine kinase binding motif. CD8a is expressed on the cell surface as either a disulfide bond-linked homodimer (CD8αα) or a heterodimer (CD8αβ) when disulfide bonded to CD8b (also known as, CD8 beta/CDβ, Ly-3, or Lyt-3), another type I transmembrane glycoprotein. CD8aβ is expressed on most thymocytes and a subpopulation of mature peripheral TCR αβ T cells including some intraepithelial lymphocytes (IELs). CD8αα is expressed on IELs which include either TCR αβ or TCR γδ T cells as well as on other leucocytes, such as, subsets of NK cells and dendritic cells (DCs). CD8αβ serves as a co-receptor for antigen-stimulated CD8+ T cells by binding to the same peptide:MHC class I complex as the T cell receptor and by helping to trigger the subsequent signaling cascade through Lck activation upon antigen recognition. Through high-affinity binding to nonclassical MHC class-Ib molecules, CD8αα can reportedly function less efficiently as a co-receptor and even repress antigen-stimulated CD8+ T cell responses.

        

567630 Rev. 1
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
567630 Rev.1
Citations & References
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Development References (10)

  1. Barclay NA, Brown MH, Birkeland ML, et al, ed. The Leukocyte Antigen FactsBook. San Diego, CA: Academic Press; 1997.
  2. Camerini V, Panwala C, Kronenberg M. Regional specialization of the mucosal immune system. Intraepithelial lymphocytes of the large intestine have a different phenotype and function than those of the small intestine.. J Immunol. 1993; 151(4):1765-76. (Biology). View Reference
  3. Goodall KJ, Nguyen A, McKenzie C, Eckle SBG, Sullivan LC, Andrews DM. The murine CD94/NKG2 ligand, Qa-1(b), is a high-affinity, functional ligand for the CD8αα homodimer. J Biol Chem. 2020; 295(10):3239-3246. (Biology). View Reference
  4. LeFrancois L. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire. Immunol Today. 1991; 12(12):436-438. (Biology). View Reference
  5. Leishman AJ, Naidenko OV, Attinger A, et al. T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL. Science. 2001; 294(5548):1848-1849. (Biology). View Reference
  6. Probst HC, McCoy K, Okazaki T, Honjo T, van den Broek M. Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4.. Nat Immunol. 2005; 6(3):280-6. (Clone-specific: Flow cytometry). View Reference
  7. Ren H, Ferguson BJ, Maluquer de Motes C, Sumner RP, Harman LE, Smith GL. Enhancement of CD8(+) T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor.. Immunology. 2015; 145(1):34-49. (Clone-specific: Flow cytometry). View Reference
  8. Sydora BC, Brossay L, Hagenbaugh A, Kronenberg M, Cheroutre H. TAP-independent selection of CD8+ intestinal intraepithelial lymphocytes. J Immunol. 1996; 156(11):4209-4216. (Biology). View Reference
  9. Takahashi K, Nakata M, Tanaka T, et al. CD4 and CD8 regulate interleukin 2 responses of T cells. Proc Natl Acad Sci U S A. 1992; 89(12):5557-5561. (Immunogen: Functional assay, Immunofluorescence, Inhibition). View Reference
  10. Vremec D, Zorbas M, Scollay R, et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med. 1992; 176(1):47-58. (Biology). View Reference
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567630 Rev. 1

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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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.