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

Clone 500A2 is a monoclonal antibody targeting mouse CD3ε and is widely used for in vivo immunological research in mice, particularly to engage or deplete T cells and to modulate the immune response.

Common in vivo applications include:

• T cell depletion: Used to deplete T cells in mice to study immune system function or to create T-cell-deficient models.
• T cell activation: Administration can activate T lymphocytes, making it useful in models examining T cell-driven immune responses, such as autoimmunity or immune tolerance induction.
• Modulating immune responses: Enables the study of immune regulation and the roles of T cells by either stimulating or suppressing their activity.
• Experimental therapeutics for T cell disorders: Applied in developing or assessing treatments for T cell-driven diseases, such as autoimmune disorders or graft-versus-host disease.
• Immunophenotyping and functional assays: Used as a marker to identify and analyze T cells during in vivo studies, typically through flow cytometry or histological assessment.

Additional context:

• Clone 500A2 specifically binds to the CD3ε chain, a component of the T cell receptor complex expressed on thymocytes, mature T cells, and NKT cells.
• The antibody can be used in various formats (soluble, plate-bound) to modulate T cell function, and it does not cross-react with rat leukocytes.
• Functional-grade, low-endotoxin preparations are preferred for in vivo use to minimize off-target or inflammatory effects.
• While it is well-established for in vivo functional depletion or modulation of T cells, it can also be used for immunophenotyping in both flow cytometry and immunohistochemistry with appropriate formulations.

In summary, clone 500A2 is primarily used for in vivo T cell depletion or activation, immune response modulation, immunophenotyping, and as a research tool to interrogate T cell-dependent processes in mouse models.

Commonly used antibodies or proteins in the literature alongside 500A2 (anti-mouse CD3ε) include other anti-CD3 clones, as well as antibodies against major lymphocyte surface markers and functional proteins. These are selected to aid in immunophenotyping, T cell activation studies, and multi-color flow cytometry assays.

Key examples regularly used with 500A2:

• Other anti-CD3 clones:

  • 17A2
  • 145-2C11
    These clones are sometimes used for comparative analysis or epitope mapping because they recognize distinct but overlapping regions on CD3ε.

• Surface marker antibodies:

  • CD4 (helper T cells)
  • CD8 (cytotoxic T cells)
  • CD14 (monocytes)
  • CD20 (B cells)
  • CD34 (hematopoietic progenitors)
    These are used to define immune cell populations and for multi-marker analysis.

• Functional antibodies for blocking or activation:

  • GK1.5 (anti-CD4): Used to block CD4 association with the TCR complex, especially in T cell activation studies.

• Secondary antibodies:

  • Typically, labeled secondaries like HRP, alkaline phosphatase, biotin, or fluorescent conjugates are employed depending on the detection method (flow cytometry, ELISA, immunohistochemistry).

• Proteins or reagents tied to T cell activation studies:

  • Plate-bound 500A2 activates T cells, so reagents for proliferation and cytokine assays (e.g., IL-2 detection kits) are common companions.

In summary, the most frequent partners for 500A2 in published protocols are other anti-CD3 clones (17A2, 145-2C11), antibodies for CD4, CD8, and other lineage markers (CD14, CD20, CD34), and detection secondaries or functional reagents for cell activation and blocking studies.

Overview of Clone 500A2 Anti-Mouse CD3ε Antibody

Clone 500A2 is a well-characterized monoclonal antibody raised in Syrian hamster that specifically recognizes mouse CD3ε, a critical component of the T cell receptor (TCR) complex. CD3ε is expressed primarily on mature T cells and NK-T cells, with varying levels on thymocytes depending on differentiation status. The TCR complex, including CD3ε, is essential for T cell signaling and immune function.

Key Scientific Findings Involving Clone 500A2

Epitope Specificity and Cross-Blocking

• Clone 500A2 recognizes a distinct but overlapping epitope compared to other anti-CD3 antibodies like 17A2 and 145-2C11, as demonstrated by partial cross-blocking in binding studies.
• This implies that all three antibodies (500A2, 17A2, 145-2C11) bind to different but closely located regions on the CD3ε molecule, which enables researchers to use them simultaneously or consecutively for staining or functional studies.

Functional and Biological Applications

• 500A2 has been used to activate mouse T cells in vitro, functioning as a potent T cell stimulator. When employed in this capacity, it is commonly used in its functional grade, low-endotoxin formulation to minimize unintended immune activation.
• The antibody effectively stains thymocytes and splenocytes for flow cytometry, typically at concentrations of ≤0.5–1.0 µg per 10^6 cells in 100 µL, and is also validated for immunohistochemistry on frozen tissue sections at 2.5–10 µg/mL, though titration is recommended for optimal results in specific assays.
• 500A2 is also used for in vivo studies to engage or deplete T cells in mouse models, supporting its role in experimental immunology as a tool for T cell manipulation.

Technical Validation

• The antibody is highly pure (>90% by SDS-PAGE), with low aggregation (<10% by HPLC) and minimal endotoxin, making it suitable for sensitive in vitro and in vivo applications.
• Multiple suppliers provide different conjugates (e.g., biotin, Alexa Fluor 647, AF700) for advanced flow cytometry and sorting applications, extending its utility in T cell phenotyping and functional analysis.

Citation Context and Usage

• Original articles (e.g., Miescher GC, et al. Immunol. Lett. 1989) and technical manuals report the use of 500A2 for T cell activation and characterization, but detailed mechanistic findings (e.g., specific signaling pathways, in vivo outcomes) are not summarized in the provided citations.
• Clone 500A2 is often used alongside other anti-CD3 antibodies in the literature to cross-validate results or to take advantage of its unique epitope specificity.
• The antibody is cited in protocols for generating induced T cells and for functional assays involving TCR engagement.

Summary Table

Conclusion

Clone 500A2 is a versatile and reliable tool for the detection, activation, and manipulation of mouse T cells, recognized for its unique epitope specificity, high purity, and broad applicability in flow cytometry, immunohistochemistry, and functional assays. Its ability to partially cross-block other anti-CD3 antibodies underscores its value in multiplexed experimental designs and mechanistic studies of T cell biology.

The dosing regimens of clone 500A2, an anti-mouse CD3ε antibody, vary significantly depending on the experimental mouse model and application. There is no universal standard, and dosing must be empirically optimized for each specific system and intended use.

Key details about dosing regimens across mouse models:

• For flow cytometry, 500A2 is typically used at concentrations of ≤1.0 µg per million cells in 100 µl volume.
• For immunohistochemistry (on frozen tissue sections), recommended concentrations range from 2.5–10 µg/ml.
• For in vivo functional or depletion studies (such as T cell activation or modulation), literature and supplier protocols highlight significant variability:
  • Leinco Technologies emphasizes that all protocols require empirical optimization with no universal regimen, reflecting heterogeneity in target mouse strains, disease models, study endpoints, and desired immunological outcomes.
  • Published studies sometimes describe protocols where 500A2 is administered intraperitoneally at doses such as 10–50 µg/mouse, but exact regimens (dose, frequency, duration) are tailored for each experiment based on parameters like T cell activation potency, toxicity, and strain-specific responses. For example, activation of T lymphocytes was achieved by injecting 50 µg intraperitoneally in C3H/HeJ mice in one model, but this regimen is specific to that experimental condition and strain.
  • The route is most often intraperitoneal or intravenous, but may also vary.

Critical context:

• Dosing is strongly influenced by application (e.g., short-term activation vs. chronic depletion), mouse strain (genetic background), and disease model (autoimmunity, infection, cancer).
• The need for "empirical optimization" means that researchers often start with doses reported in similar prior studies but perform titrations for their specific system to balance efficacy and toxicity.
• There is overlap in the use of 500A2 with other anti-CD3 clones (like 145-2C11 or 17A2), but each clone may have distinct dosing characteristics and cross-reactivities.

Summary Table:

Conclusion:
Researchers must adjust 500A2 dosing protocols to match the requirements of each mouse model and experimental objective; reliance on literature precedents is common, but pilot titration studies are universally advised due to the absence of an established dosing standard.