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Mouse Monoclonal to CD3 activation epitope

APA1/1 (IgG1)

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Background

CD3 complex is crucial in transducing antigen-recognition signals into the cytoplasm of T cells and in regulating the cell surface expression of the TCR complex. T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3 gamma, CD3 delta, CD3 epsilon and CD3 zeta. These CD3 subunits are structurally related members of the immunoglobulins super family encoded by closely linked genes on human chromosome 11. The CD3 components have long cytoplasmic tails that associate with cytoplasmic signal transduction molecules. This association is mediated at least in part by a double tyrosine-based motif present in a single copy in the CD3 subunits. CD3 may play a role in TCR-induced growth arrest, cell survival and proliferation.

Specificity

The mouse monoclonal antibody APA1/1 recognizes an activation-dependent intracellular epitope of CD3 epsilon. Exposure of the epitope precedes CD3 phosphorylation and recruitment and activation of ZAP70, which initiates the signaling cascade produced by T-cell activation. APA1/1 provides the earliest known marker for TCR-mediated T cell activation.

Regulatory Status

Immunogen

Purified human CD3 proteins isolated from thymus

Species Reactivity:

  • Human
  • Mouse

Negative Species:

Applications:

  • Flow Cytometry
    Positive control: Human T cells stimulated with anti-CD3 (MEM-57) antibody (1 μg/ml)
    Sample preparation:
    At the end of stimulation of T cells, perform staining of surface markers (if required) in PBS + 0.1% BSA for 20 min. on ice.
    Wash with PBS and fix with 2% formaldehyde, 30 min on ice.
    Wash with PBS and incubate in PBS + 0,1% saponine, 5 min. RT.
    Incubate the cells in PBS + 1% BSA + 0.03% saponine, 15 min. on ice.
    Incubate with fluorescence-labeled APA1/1 antibody (1-2 μg/ml) in PBS + 1% BSA + 0.03% saponine in dark, 20 min. RT.
    Wash with PBS + 1% BSA + 0.03% saponine, resuspend in PBS.
  • Immunoprecipitation
  • Western Blotting
  • Immunohistochemistry (frozen sections)
  • Immunocytochemistry
    Application note:Fixed and permeabilised cells. The antibody can distinguish TCR-stimulated from non-stimulated cells.
Usage note:
Indicated dilutions are recommended starting points for use of this product. Working concentrations should be determined by the investigator.

Product Specific References

  • *Alarcón B, Ley SC, Sánchez-Madrid F, Blumberg RS, Ju ST, Fresno M, Terhorst C: The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes. EMBO J. 1991 Apr;10(4):903-12. [Abstract] [Full Text]
  • *Wong AP, Dutly AE, Sacher A, Lee H, Hwang DM, Liu M, Keshavjee S, Hu J, Waddell TK: Targeted cell replacement with bone marrow cells for airway epithelial regeneration. Am J Physiol Lung Cell Mol Physiol. 2007 Sep;293(3):L740-52. [Abstract] [Full Text]
  • *Torres PS, Alcover A, Zapata DA, Arnaud J, Pacheco A, Martín-Fernández JM, Villasevil EM, Sanal O, Regueiro JR: TCR dynamics in human mature T lymphocytes lacking CD3 gamma. J Immunol. 2003 Jun 15;170(12):5947-55. [Abstract] [Full Text]
  • *Rieux-Laucat F, Hivroz C, Lim A, Mateo V, Pellier I, Selz F, Fischer A, Le Deist F: Inherited and somatic CD3zeta mutations in a patient with T-cell deficiency. N Engl J Med. 2006 May 4;354(18):1913-21. [Abstract] [Full Text]
  • *Tailor P, Tsai S, Shameli A, Serra P, Wang J, Robbins S, Nagata M, Szymczak-Workman AL, Vignali DA, Santamaria P: The proline-rich sequence of CD3epsilon as an amplifier of low-avidity TCR signaling. J Immunol. 2008 Jul 1;181(1):243-55. [Abstract] [Full Text]
  • *Delgado P, Fernández E, Dave V, Kappes D, Alarcón B: CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection. Nature. 2000 Jul 27;406(6794):426-30. [Abstract]
  • *Risueño RM, Gil D, Fernández E, Sánchez-Madrid F, Alarcón B: Ligand-induced conformational change in the T-cell receptor associated with productive immune synapses. Blood. 2005 Jul 15;106(2):601-8. [Abstract] [Full Text]
  • *Gil D, Schrum AG, Alarcón B, Palmer E: T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes. J Exp Med. 2005 Feb 21;201(4):517-22. [Abstract] [Full Text]
  • *Risueño RM, van Santen HM, Alarcón B: A conformational change senses the strength of T cell receptor-ligand interaction during thymic selection. Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9625-30. [Abstract] [Full Text]
  • *And other.
  • For research use only. Not for drug, diagnostic or other use.

    Example Data

    APA1-1 pur


    Fig. 1. Intracellular staining of human peripheral blood with anti-CD3 epsilon activation epitope (APA1/1) purified, GAM-FITC.


    APA1-1 PE


    Fig. 2. Intracellular staining of JURKAT cells with anti-CD3 epsilon activation epitope (APA1/1) PE.

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