Anti-Mouse CD3ε [Clone 500A2] — Purified in vivo GOLD™ Functional Grade

Clone 500A2 is a monoclonal antibody that recognizes the mouse CD3? chain, and it is commonly used in in vivo mouse studies to engage or deplete T cells, modulate immune responses, or as a marker in immunophenotyping and functional assays. Its main uses in vivo fall into these categories:

• T Cell Activation/Functional Studies: 500A2 targets CD3?, a component of the T cell receptor (TCR) complex critical for signal transduction leading to T cell activation. In vivo administration of anti-CD3? (such as 500A2) can mimic antigen engagement, leading to polyclonal T cell activation and proliferation. Depending on dose and context, this can promote T cell expansion or induce activation-induced cell death or immune modulation.

• Immunomodulation and Tolerance Induction: High doses of anti-CD3 antibodies like 500A2 are sometimes used to induce transient T cell depletion or reversible immunosuppression, which has been leveraged in models of autoimmune disease, transplantation tolerance, or immune resetting studies. This effect is often transient, and the specific depletion or modulation profile can vary with dose and experimental protocol.

• Enumeration and Depletion of T Cells: 500A2 can be used to quantitatively and qualitatively assess T cell populations in vivo, often via flow cytometry using cells extracted from treated animals. At higher or sustained doses, it may lead to partial depletion of peripheral T cells or modulation of thymocytes.

• Immunophenotyping: Following in vivo administration, 500A2 can be used ex vivo to stain tissue or blood samples for CD3+ cells, analyzing effects on T cell subsets or monitoring immune reconstitution.

Typical protocols and notes:

• The antibody is Syrian Hamster IgG specific for mouse, verified for in vivo and in vitro use.
• Recommended dosing and application specifics depend on the objective (activation, depletion, or tracking), and the optimal concentration and regimen must be empirically determined for each experimental setup.
• In some protocols, cross-blocking capability with other anti-CD3 clones (e.g., 17A2, 145-2C11) is leveraged for mechanistic or combinatorial studies.
• 500A2 can be conjugated with various fluorophores or labels for downstream flow cytometry or imaging.

Key properties:

• Recognizes the CD3? subunit (20–25?kDa), expressed on all mature T cells, thymocytes, and NKT cells.
• Works for flow cytometry (FC), immunohistochemistry (IHC-F), cell function/blocking studies, and in some cases for T cell depletion in vivo.

Summary Table:

For precise usage—such as dosing, timing, or combination with other agents—consult relevant publications and adapt based on the specific experimental goal.

If you need a protocol for a specific experimental goal (e.g., T cell depletion, activation, disease model), please clarify, as some protocols vary significantly depending on study design.

The correct storage temperature for the sterile packaged clone 500A2 (anti-mouse CD3? antibody) is between 2°C and 8°C undiluted, and it should not be frozen.

According to multiple sources, including BioLegend and BD Biosciences, the antibody solution must be kept undiluted at refrigerator temperatures (2–8°C) and protected from prolonged exposure to light. Syd Labs further specifies that, as supplied, the antibody is stable for 12 months at 2–8°C and should not be frozen.

If your product is a different formulation or specifically designed for long-term frozen storage, always verify with the supplier’s specific datasheet. However, for the standard sterile packaged research antibody, 2–8°C storage is correct.

Commonly Used Antibodies and Proteins with 500A2 in the Literature

The 500A2 antibody is a monoclonal antibody specific for the CD3? chain of the mouse T-cell receptor (TCR) complex and is widely used in immunology research, particularly for flow cytometry and immunohistochemistry (IHC) of mouse tissues. While 500A2 directly recognizes the CD3? molecule, its use is often combined with other reagents for multiparametric analysis or functional studies.

Frequently Paired Antibodies and Proteins

• Anti-CD4 mAb (clone RM4-5): Commonly used in conjunction with 500A2 for simultaneous staining of mouse splenocytes to distinguish CD4? T cells from other lymphocyte populations; RM4-5 is typically conjugated to PE for fluorescence detection.
• Anti-CD8a mAb (clone 53-6.7): Frequently used alongside 500A2 and anti-CD4 to identify and separate CD8? T cells; 53-6.7 is also often PE-conjugated.
• Anti-Hamster IgG mAb (clone G192-1, FITC-conjugated): Since 500A2 is a hamster antibody, a fluorescently labeled anti-hamster secondary antibody (e.g., G192-1-FITC) is commonly used to detect 500A2 binding by flow cytometry.
• GK1.5 (anti-CD4 Fab fragments): Used in functional studies to block T-cell activation mediated by plate-bound 500A2, indicating the proximity of the 500A2 epitope to the CD4-TCR interaction site.
• Other anti-CD3 Antibodies (e.g., 17A2, 145-2C11): 500A2 has been shown to partially cross-block these antibodies, suggesting they recognize overlapping but distinct epitopes on CD3?. These clones are sometimes used in comparative or epitope-mapping studies.

Typical Experimental Contexts

• Multicolor Flow Cytometry: 500A2 (detected via anti-hamster secondary) is combined with directly conjugated anti-CD4 (RM4-5-PE) and anti-CD8a (53-6.7-PE) to phenotype T-cell subsets in mouse lymphoid organs.
• Functional T-Cell Assays: Plate-bound 500A2 is used to activate T cells in vitro, with GK1.5 Fab fragments used to probe the role of CD4 in this activation.
• Comparative Epitope Studies: 500A2 is sometimes used alongside other anti-CD3? antibodies (e.g., 17A2, 145-2C11) to map epitopes or assess antibody cross-reactivity.

Summary Table

Additional Considerations

• Isotype Controls: Non-specific hamster IgG antibodies may be used as negative controls in experiments involving 500A2.
• Lymphocyte Activation Proteins: Cytokines (e.g., IL-2) or co-stimulatory molecules (e.g., CD28) may be used in functional assays where 500A2 is the primary stimulus, but these are not antibodies.
• Species Specificity: 500A2 does not cross-react with rat leukocytes, so its use is generally limited to mouse samples.

In summary, anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-hamster IgG (G192-1-FITC), and anti-CD3? clones 17A2 and 145-2C11 are among the most commonly used antibodies in combination with 500A2 in the literature, primarily for phenotypic and functional studies of mouse T cells.

Based on scientific literature, clone 500A2 is a well-characterized monoclonal antibody with several key findings and applications documented in research studies.

Antibody Characteristics and Specificity

Clone 500A2 is a Syrian hamster IgG2a ? monoclonal antibody that specifically recognizes an epitope on mouse CD3?. This antibody has been instrumental in T-cell research due to its high specificity for the CD3? subunit, which is a 20kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily.

Target Protein Function and Distribution

The CD3? protein targeted by clone 500A2 serves critical functions in T-cell biology. It forms part of the T-cell receptor (TCR) complex by associating with CD3?, ?, and ? chains, along with TCR ?/? or ?/? chains. This complex is essential for TCR signaling and plays a vital role in signal transduction during antigen recognition. The protein is primarily expressed on mature T cells and NK-T cells, with varying expression levels on differentiation-dependent thymocytes.

Research Applications

Clone 500A2 has proven valuable in multiple research contexts. It is widely used in flow cytometry applications for enumerating immunocompetent T-lymphocytes in peripheral blood and analyzing T-cell populations. The antibody has been particularly useful in studies involving T-cell reprogramming, as demonstrated in protocols for generating functional T cells from B-cell precursors.

Historical Significance and Citations

The antibody has substantial historical significance in immunological research, with foundational citations dating back to the late 1980s. Key early publications include work by Havran et al. (1987) published in Nature (volume 330:170) and studies by Allison et al. (1988) in "The T-Cell Receptor". These early studies established the antibody's utility and contributed to our understanding of T-cell receptor biology.

Technical Applications

For practical applications, clone 500A2 is recommended for use at concentrations of ?1.0 ?g per 10? cells in a 100 ?l volume for flow cytometry staining. The antibody is available in various formats including purified and biotinylated forms, making it versatile for different experimental approaches. Its consistent performance has made it a standard tool in immunology laboratories for T-cell identification and functional studies.

The extensive citation history and continued use of clone 500A2 in contemporary research, including advanced applications like spectral flow cytometry for metabolic profiling, demonstrates its enduring value in the scientific community.