Anti-Mouse CD8a (Ly 2) [Clone 53-6.7] — Purified in vivo GOLD™ Functional Grade

Clone 53-6.7 is primarily used in in vivo mouse studies for depletion of CD8a+ cells (cytotoxic T lymphocytes), blocking antigen presentation via MHC class I, and inhibiting T cell responses such as IL-2 production. This antibody binds to the CD8a molecule found on cytotoxic T cells, certain thymocytes, as well as NK cells and dendritic cells in mice.

Essential applications in in vivo mouse studies include:

• Depletion of CD8a+ cells: Used to physically remove or ablate cytotoxic T cells from mice via antibody-mediated clearance, to study immune function or disease mechanisms without CD8a+ T cells.
• Functional blocking: Interferes with the interaction of CD8a with MHC class I, thereby inhibiting antigen presentation and downstream T cell activation and proliferation (for example, blocking IL-2 responses or cytotoxicity assays).
• Phenotypic identification: Used as a marker to identify and analyze CD8a+ cells in vivo by flow cytometry, which is critical for immunophenotyping in disease and immune response models.

Additional reported uses include:

• Immunoprecipitation and immunohistochemical staining of tissue sections (not recommended for formalin-fixed sections).
• Competition assays where clone 53-6.7 can block other anti-CD8a clones (such as 5H10-1).
• Studies of spatial biology, e.g., IBEX multiplexed tissue imaging.

For in vivo studies, purified or ultra-low endotoxin preparations (e.g., Ultra-LEAF™) are recommended to minimize unwanted inflammation.

In summary, clone 53-6.7 is a key tool for depleting, functionally blocking, or identifying CD8a+ cells in live mouse models, to dissect their role in immunity, infection, cancer, or autoimmune disease.

Commonly used antibodies and proteins paired with 53-6.7 (anti-mouse CD8a) in the literature include markers and functional antibodies relevant for immune cell phenotyping and functional assays in murine models.

Frequently co-used antibodies/proteins:

• CD4 Antibodies: Used to distinguish helper T cells (CD4+) from cytotoxic T cells (CD8+), commonly in flow cytometry and tissue analysis.
• CD3 Antibodies: Pan-T cell marker, often included for comprehensive T cell profiling.
• CD45 Antibodies: Leukocyte common antigen used to identify all hematopoietic cells.
• Isotype Controls: Such as purified rat IgG2a, ? isotype control (e.g., clone 2A3), matched for antibody subclass and species to control for background staining.
• CD8b Antibodies: Used in studies requiring distinction between CD8a and CD8b chains, especially when dissecting T cell subsets.
• Cytokine Detection Antibodies: For intracellular staining and functional assessment (e.g., IL-2, IFN-?), sometimes in assays where CD8+ T cell effector function is measured.
• Viability Dyes or Exclusion Dyes: To distinguish live/dead cells in flow cytometry panels.

Context and applications:

• 53-6.7 is specifically designed to bind mouse CD8a (expressed on cytotoxic T cells), making it a standard antibody in murine immunophenotyping, in vivo depletion, blocking, immunoprecipitation, and immunohistochemistry in mouse tissues.
• Studies frequently use CD8a (53-6.7) together with markers for T cell subsets (CD4, CD3) or with other lineage-specific markers and functional antibodies to analyze immune responses and T cell biology in mice.
• For depletion or blocking studies, function-blocking antibodies against molecules such as CD4, CD8b, or major histocompatibility complex (MHC) proteins may also be included in parallel with 53-6.7.

Summary Table: Commonly Co-Used Antibodies/Proteins with 53-6.7

These combinations enable detailed characterization and functional studies of murine T cell populations in immunological research.

The clone 53-6.7 antibody, which targets mouse CD8a, has generated several important findings in the scientific literature that have established it as a valuable research tool for studying T cell biology and immune responses.

Functional Properties and Mechanisms

The 53-6.7 antibody demonstrates significant blocking activity against key immune processes. It has been shown to block antigen presentation via MHC class I molecules and inhibit T cell responses to IL-2. These properties make it particularly useful for studying the mechanisms of T cell activation and antigen recognition.

The antibody also exhibits competitive binding characteristics, as it competes with clone 5H10-1 antibody for binding to thymocytes. This finding suggests that both antibodies recognize similar or overlapping epitopes on the CD8a molecule.

Target Specificity and Expression

Research has established that the 53-6.7 monoclonal antibody specifically binds to the 38 kDa ? and 34 kDa ?' chains of the CD8 differentiation antigen across all mouse strains tested. The CD8a chains form heterodimers with the CD8? chain on most thymocytes, while mature T lymphocytes, particularly MHC class I-restricted T cells including cytotoxic T cells, express predominantly the CD8 ?? heterodimer.

Importantly, certain cell populations express CD8a without CD8b, including subsets of ?? TCR-bearing T cells, intestinal intraepithelial lymphocytes, and dendritic cells. This expression pattern has led to the suggestion that CD8a/CD8b heterodimer expression is restricted to T lymphocytes that matured in the thymus or in extrathymic environments influenced by thymus-initiated neuroendocrine signals.

Experimental Applications

The literature has documented numerous research applications for clone 53-6.7. These include immunoprecipitation, both in vivo and in vitro cell depletion studies, inhibition of CD8 T cell proliferation, and blocking of cytotoxicity. The antibody has also proven effective for immunohistochemical staining of acetone-fixed frozen sections and zinc-fixed paraffin-embedded sections, as well as spatial biology applications using IBEX methodology.

However, an important limitation has been identified: clone 53-6.7 is not recommended for immunohistochemistry of formalin-fixed paraffin sections, which restricts its use in certain histological applications.

Therapeutic and Depletion Studies

The antibody has been successfully used for depletion of CD8a+ cells in experimental settings, making it valuable for studies investigating the role of CD8+ T cells in various disease models and immune responses. For functional assays and in vivo studies, specialized formulations with ultra-low endotoxin levels have been developed to ensure reliable results without confounding inflammatory effects.

These findings collectively establish clone 53-6.7 as a well-characterized and versatile tool for CD8a research, with documented efficacy across multiple experimental approaches while noting specific technical limitations that researchers should consider when designing experiments.

Dosing regimens for clone 53-6.7—a rat anti-mouse CD8? monoclonal antibody—vary across different mouse models depending on factors such as experimental objective, mouse strain, route of administration, and whether acute depletion or chronic suppression is needed.

• Standard Dose and Route: In typical immunology studies with common strains (e.g., C57BL/6, BALB/c), 53-6.7 is generally administered by intraperitoneal (i.p.) or intravenous (i.v.) injection at doses ranging from 100 ?g to 500 ?g per mouse per injection. Doses around 200–250 ?g per mouse i.p. are commonly used for effective CD8+ T cell depletion.

• Frequency: Injection schedules generally involve a single initial "depleting" dose followed by maintenance doses every 3–7 days, depending on study length and requirements for sustained depletion. Some protocols favor a single high dose followed by smaller, less frequent maintenance doses for chronic studies.

• Model-Dependent Adjustments:

  • In standard tumor or infection models (e.g., transplantation, viral challenge), the 250 ?g per dose schedule is widely effective.
  • For transgenic, immunodeficient, or aged mice, dosing might be adjusted—either increased (for higher body mass or reduced sensitivity) or decreased (to mitigate toxicity or avoid excessive immunosuppression).
  • In neonatal or particularly sensitive models, lower doses (e.g., 100 ?g per mouse) or increased intervals between injections may be used for safety.

• Comparison with Other Clones: Like other CD8?-depleting clones (e.g., 2.43, YTS 169.4), the effective range for 53-6.7 is broadly similar, but specific optimization for model and endpoint is common. For humanized or rat models, other clones (e.g., OKT8, H35-17.2) are used.

• Reported Biological Effects: Clone 53-6.7 blocks antigen presentation via MHC class I and is documented to inhibit T cell responses to IL-2 as well as to deplete CD8?+ cells efficiently in vivo.

• Toxicity: At standard doses (?500 ?g/mouse), specific toxicity is minimal in most mouse models; higher or repetitive dosing may increase the risk for immunogenicity or systemic effects, especially in chronic administration.

In summary, clone 53-6.7 is typically used at 100–500 ?g per mouse (often 200–250 ?g), with dosing frequency varying from a single dose to weekly maintenance, across a range of mouse models, and protocols are routinely tailored for strain, experimental endpoint, and sensitivity.