CD3 Monoclonal Antibody (OKT3), NovaFluor™ Yellow 590, eBioscience™

Product Specific Information

Description
The OKT3 monoclonal antibody reacts with an epitope on the epsilon-subunit within the human CD3 complex. The OKT3 antibody has been reported to have potent immunosuppressive properties in vivo and has been proven effective in the treatment of renal, heart and liver allograft rejection. Antibody clones OKT3 and SK7 see different epitopes.

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

Applications Tested
This OKT3 antibody has been pre-diluted and tested by flow cytometric analysis of normal human peripheral blood cells. This may be used at 5 µL (0.2 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.

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 CD3 complex, composed of gamma, delta, epsilon, and zeta subunits, is essential for the assembly, trafficking, and surface expression of the T cell receptor (TCR) complex. These subunits are structurally related members of the immunoglobulin superfamily and are encoded by closely linked genes on human chromosome 11. CD3 is expressed by thymocytes in a developmentally regulated manner and by all mature T cells, but not on B or NK cells. The CD3 subunits play a crucial role in transducing antigen-recognition signals into the cytoplasm of T cells. The cytoplasmic tails of CD3 subunits contain a double tyrosine-based motif that associates with cytoplasmic signal transduction molecules, mediating T cell activation through the TCR. Crosslinking of the TCR initiates intracellular biochemical pathways that result in cellular activation, proliferation, and potentially growth arrest and cell survival. CD3 is present on 68-82% of normal peripheral blood lymphocytes, 65-85% of thymocytes, and Purkinje cells in the cerebellum. Decreased percentages of T lymphocytes may be observed in some autoimmune diseases. Defects in the CD3 gene are associated with CD3 immunodeficiency, highlighting its importance in immune function and regulation.