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

Clone 500A2 is a hamster monoclonal antibody that specifically targets the CD3? (epsilon) chain of the mouse T-cell receptor/CD3 complex and is widely used in in vivo mouse studies to modulate or analyze T-cell activity.

Key in vivo applications of 500A2 include:

• T cell activation or depletion: When administered in vivo (injected into mice), 500A2 can bind to CD3? on surface of T cells, leading to T-cell activation, depletion, or immune modulation depending on dose and context. This property is essential for research exploring immune response, T cell dependencies in disease models, and evaluating immunotherapeutic approaches.
• Functional studies: The antibody is often used to trigger or block T-cell responses directly in living mice, thus enabling the study of T-cell-mediated immune mechanisms in various pathological or physiological settings.
• Control and mechanistic studies: In infection, autoimmunity, cancer, or transplantation models, 500A2 can be used to alter T-cell populations in mice, enabling researchers to dissect the role of T lymphocytes in these settings.

Product considerations for in vivo use:

• The in vivo grade antibody is supplied with ultra-low endotoxin content to minimize non-specific immune activation.
• The recommended storage is at 2–8°C and the antibody should not be frozen.
• Applications include not only in vivo blocking/depletion, but also immunohistochemistry (IHC), flow cytometry (FC), and similar.

Epitopic specificity and mechanism:

• Clone 500A2 binds a defined epitope on mouse CD3?, which is essential for TCR-mediated signaling. Its binding can either mimic or block natural TCR engagement, depending on presentation (soluble vs. plate-bound) and dosage.
• This antibody does not cross-react with rat leukocytes.

Summary table:

In summary, clone 500A2 is primarily used in vivo for targeted T-cell modulation—depletion, activation, or functional studies—by binding to CD3? on mouse T cells. It is essential to use an endotoxin-free, validated in vivo grade preparation for these studies.

Commonly used antibodies or proteins alongside 500A2 (anti-mouse CD3?) in the literature include other anti-CD3 clones such as 17A2 and 145-2C11, as well as antibodies targeting surface markers like CD4, CD8, CD14, CD20, and CD34.

Researchers frequently pair 500A2 with:

• 17A2 and 145-2C11 (other anti-CD3 clones), as 500A2 partially cross-blocks these antibodies, indicating overlapping but distinct epitope recognition on CD3?.
• CD4 and CD8 antibodies to distinguish and analyze T cell subsets in flow cytometry or functional assays.
• Antibodies against markers such as CD14 (monocytes), CD20 (B cells), and CD34 (hematopoietic progenitor cells) for multiparametric immunophenotyping in research and diagnostic settings.
• Fluorescently conjugated antibodies for detection in applications such as flow cytometry (FC) and immunohistochemistry (IHC).

In addition to cell surface markers:

• Antibodies against cytokines (e.g., TNF, IL-6) and checkpoint proteins (e.g., PD-1) are common in functional studies and therapeutic research, though less directly paired with CD3 antibodies in standard immunophenotyping workflows.

These combinations are used across platforms like flow cytometry, ELISA, immunofluorescence, and immunohistochemistry to enable detailed analysis of immune cell populations and activation states.

Key Scientific Findings on Clone 500A2

Epitope and Specificity

• Clone 500A2 is a monoclonal antibody that specifically binds the 25-kDa ? chain (CD3?) of the mouse T-cell receptor (TCR)-associated CD3 complex.
• Its target, CD3?, is expressed on mouse thymocytes, mature T lymphocytes, and NKT cells.
• CD3? is a transmembrane glycoprotein and a critical subunit of the TCR complex, essential for TCR signaling, T-cell enumeration, and antigen recognition.

Functional Characterization

• Activation of T Cells: Both plate-bound and soluble forms of the 500A2 antibody can activate mouse T cells in vitro. This suggests that the antibody can mimic antigenic stimulation through the TCR complex.
• Epitope Mapping: Activation of T cells by 500A2 can be blocked by Fab fragments of the anti-CD4 antibody GK1.5, implying that the 500A2 epitope is located near the CD4 binding site on the TCR complex, possibly influencing the interaction between CD4 and the TCR.
• Cross-blocking Studies: Clone 500A2 partially cross-blocks the binding of other anti-CD3 antibodies (17A2 and 145-2C11), indicating that these antibodies recognize distinct but overlapping epitopes on CD3?.

Species Specificity and Applications

• Species Reactivity: Clone 500A2 does not cross-react with rat leukocytes, underscoring its specificity for mouse CD3?.
• Versatile Applications: The antibody is routinely validated for flow cytometry, and other applications such as immunohistochemistry have been reported in the literature. It has also been adapted for single-cell 3' sequencing applications.

Immunological Insights

• Structure-Function Relationship: The proximity of the 500A2 epitope to the CD4 binding site highlights the importance of CD3? in the spatial organization and signaling capacity of the TCR complex.
• Research Utility: Clone 500A2 is widely used as a tool to study T-cell activation, TCR signaling, and T-cell development in mouse models.

Summary Table

Conclusion

Clone 500A2 is a critical reagent for mouse immunological research, providing insights into TCR structure, T-cell activation, and the spatial relationship between CD3? and CD4. Its ability to activate T cells and its epitope specificity make it a valuable tool for studying T-cell biology and signal transduction.

Clone 500A2 dosing regimens in mouse models vary according to application (e.g., flow cytometry, immunohistochemistry, or in vivo functional studies), tissue type, and experimental aims. For most applications, optimization by titration is recommended to suit specific mouse strains and experimental protocols.

• For flow cytometry, the typical dose is ?1.0??g per million cells in 100??l volume.
• For immunohistochemical staining (IHC) on frozen tissue sections, recommended concentrations are 2.5–10.0??g/ml.
• For in vivo functional assays (such as T cell engagement or depletion in various disease or immune models), manufacturers recommend that each investigator determine optimal working dilutions via titration for their specific application and mouse model.

Additional details:

• The recommended regimens above are general guidelines and may require adjustment for factors such as mouse strain, disease context (e.g., autoimmune, infectious, or cancer models), or endpoint (activation vs. depletion).
• There is no universal dosing schedule for clone 500A2 across all mouse models due to differences in experimental design, route of administration, and desired immune modulation—researchers are advised to consult lot-specific datasheets and titrate for optimal effect in each study.

In summary:

• Flow cytometry: ?1.0??g/10? cells
• IHC (frozen): 2.5–10??g/ml
• In vivo functional: titrate based on investigator protocol and mouse model

No published standard dosing interval or specific regimen for in vivo administration of clone 500A2 in mouse models was found in these sources; researchers are expected to titrate to best fit their application.