TCR alpha/beta Monoclonal Antibody (IP26), NovaFluor™ Yellow 590, eBioscience™

Product Specific Information

Description
The IP26 monoclonal antibody reacts with the alpha beta chain of human TCR.The alpha beta TCR is expressed by the majority of peripheral T cells.

This product contains 1 vial of NovaFluor conjugate and 1 vial of CellBlox Plus Blocking Buffer.

Applications Tested
This IP26 antibody has been pre-titrated and tested by flow cytometric analysis of human peripheral blood cells. This can be used at 5 µL (0.8 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Staining with the IP26 antibody can be obstructed when OKT3 (anti-human CD3) antibody is used as a co-stain.

Master mixes
• Master mixes of NFs should be made at 2-8 °C and may be made up to 4 hours ahead of time.
• We do not recommend storing master mixes containing NovaFluor conjugates overnight or longer.

Whole Blood compatibility
• When utilizing whole blood (as opposed to density-gradient-purified PBMC), we recommend lysing red blood cells in bulk prior to staining with NovaFluor conjugates.
• See the Bulk Lysis of Human Whole Blood protocol here.
• Staining of whole blood with NovaFluor conjugates followed by lysis of red blood cells may result in higher-than-expected background staining.

Viability dye compatibility
• NovaFluor dyes are not compatible with DNA intercalating viability dyes.
• Do not use viability dyes such as propidium iodide, 7-actinomycin D (7-AAD) and DAPI. Invitrogen LIVE/DEAD Fixable Dead Cell stains are recommended for use with NovaFluor dyes.

CellBlox Plus Blocking Buffer
• This NovaFluor conjugate comes with CellBlox Plus Blocking Buffer (for 25 µg or 25 test products, Cat. No. C001T02F01; for 100 µg or 100 test, Cat. No. C001T03F01), essential for optimal staining.
• Use CellBlox Plus Blocking Buffer in all experiments with NovaFluor conjugates.
• Add 5 µL per sample to antibody cocktails/master mixes (regardless of how many Novafluor-conjugated antibodies are present) before combining with cells.
• CellBlox Plus Blocking Buffer is compatible with either Super Bright Complete Blocking Buffer or Brilliant Stain Buffer and can be used in antibody cocktails/master mixes with those reagents.
• For single-color controls, use 5 µL of CellBlox Plus Blocking Buffer per 100 µL of cell sample (10^3 to 10^8 cells).

NovaFluor conjugates are based on Phiton technology utilizing novel fluorophore-containing nucleic acid dye structures that allow for engineered fluorescent signatures with consideration for spillover and spread impacts. Learn more

Our internal testing shows that NovaFluor Yellow 590 non-specifically stains B cells in SJL mice. Non-specific staining has not been observed in BALB/c or C57BL/6 mice. Other strains have not been tested. See the Antibody Testing Data for an example of this strain-dependent difference.

Excitation: 563 nm; Emission: 594 nm; Laser: 561 nm (Yellow) Laser

Target Information

The T cell antigen receptor (TCR) consists of a ligand-specific alpha/beta heterodimer non-covalently associated with five invariant chains including the CD3 gamma/delta/eta and zeta subunits, all of which are required for efficient surface expression. T cell activation through the TCR induces cellular differentiation and/or proliferation and the production of lymphokines and cytokines. Both the CD3 and TCR zeta subunits are proposed to be responsible for the intracellular signal transduction events. Majority of T cells present in the blood, lymph and secondary lymphoid organs express TCR alpha/beta heterodimers, whereas the T cells expressing TCR gamma/delta heterodimers are localized mainly in epithelial tissues and at the sites of infection. The subunits of TCR heterodimers are covalently bonded and associate with the CD3 subunits in the endoplasmic reticulum to form functional TCR-CD3 complex. Lack of expression of any of the chains is sufficient to stop cell surface expression. The ability of T cell receptors (TCR) to discriminate foreign from self-peptides presented by major histocompatibility complex (MHC) class II molecules is essential for an effective adaptive immune response. TCR recognition of self-peptides has been linked to autoimmune disease. Mutant self-peptides have been associated with tumors. Engagement of TCRs by a family of bacterial toxins know as superantigens has been responsible for toxic shock syndrome. Autoantibodies to V beta segments of T cell receptors have been isolated from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The autoantibodies block TH1-mediated inflammatory auto-destructive reactions and are believed to be a method by which the immune system compensates for disease (ref5). T Cell and TCR Diversity Most human T cells express the TCR alpha-beta and either CD4 or CD8 molecule (single positive, SP). A small number of T cells lack both CD4 and CD8 (double negative, DN). Increased percentages of alpha-beta DN T cells have been identified in some autoimmune and immunodeficiency disorders. Gamma-delta T cells are primarily found within the epithelium. They show less TCR diversity and recognize antigens differently than alpha-beta T cells. Subsets of gamma-delta T cells have shown antitumor and immunoregulatory activity.