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

Clone 53-6.7 is a monoclonal antibody that targets mouse CD8? and is widely used in in vivo mouse studies to deplete or functionally block CD8?+ cells, identify cytotoxic T cells, and modulate immune responses.

Key in vivo applications include:

• Depletion of CD8?+ cells: Administration of 53-6.7 depletes CD8+ T cells (and other CD8?-expressing cells such as some NK cells), allowing researchers to study immune functions in their absence.
• Functional blockade: The antibody inhibits CD8 T cell responses by blocking antigen presentation via MHC class I, and thus can be used to suppress cytotoxic T cell activity and study the effects of CD8+ T cell inhibition in disease or infection models.
• Phenotypic marker: 53-6.7 is also used to identify and track CD8?-expressing T cells in vivo, owing to its high specificity.
• Blocking cytotoxic activity and proliferation: The antibody can inhibit T cell proliferation and the cytotoxic activity typically mediated by CD8+ T cells, making it essential for dissecting these T cell functions in vivo.

The Ultra-LEAF™ purified (endotoxin < 0.01 EU/µg, azide-free, 0.2 µm filtered) format is specifically recommended for in vivo studies to minimize toxicity and off-target effects.

In summary, clone 53-6.7 is crucial in mouse models for:

• Depleting or functionally blocking CD8+ T cells
• Assessing the role of cytotoxic T cells in immune responses
• Investigating CD8-dependent mechanisms in infection, cancer, and autoimmunity

Researchers commonly use 53-6.7 (anti-mouse CD8a) alongside several other antibodies and proteins to interrogate immune system components in mice. The main companion markers and antibodies used in the literature include:

• CD3: Identifies total T cells.
• CD4: Marks helper T cells to distinguish them from cytotoxic CD8+ cells.
• CD45: Pan-leukocyte marker for immune cells.
• CD44, CD62L: Activation and memory status of T cells.
• Isotype Controls: Such as purified Rat IgG2a, ? Isotype Control (clone 2A3), for assay validation.
• Other clones targeting CD8a: For example, 5H10-1 (competes with 53-6.7 for binding), used in select studies for validation and comparative analysis.

Researchers frequently combine 53-6.7 with these reagents in applications such as flow cytometry, immunohistochemistry, immunoprecipitation, cell depletion, and functional assays—to analyze and sort specific T cell populations, monitor activation and differentiation status, or perform spatial mapping in tissue sections.

In summary, CD3, CD4, and pan-leukocyte markers like CD45 are the most commonly used proteins or antibodies with 53-6.7, with isotype controls and other activation/memory markers routinely incorporated depending on experimental design.

Clone 53-6.7 is a rat monoclonal antibody that targets the mouse CD8a antigen, widely cited in immunology for its roles in identifying, depleting, and functionally blocking CD8+ T cells in both in vivo and in vitro experimental systems.

Key findings from scientific literature using clone 53-6.7 include:

• Specificity: The antibody binds the CD8 ? (38 kDa) and ?' (34 kDa) chains (Ly-2 or Lyt-2) of the CD8 differentiation antigen found in all mouse strains tested.
• Functional Blocking: 53-6.7 can block antigen presentation via MHC class I and inhibit CD8+ T-cell responses to IL-2, making it a tool for studying antigen-specific cytotoxic T cell function.
• Cell Depletion: It is commonly used for depleting CD8a+ cells, both in vivo (whole animal) and in vitro (cell culture), allowing researchers to study immune responses in the absence of cytotoxic T cells.
• Competition and Recognition: 53-6.7 competes with other clones (notably 5H10-1) for binding to thymocytes, confirming its binding epitope specificity.
• Immunoprecipitation and Immunohistochemistry: The antibody is validated for immunoprecipitation and immunohistochemical staining (acetone-fixed frozen and zinc-fixed paraffin sections, but not formalin-fixed paraffin sections).
• Blocking Cytotoxicity and Proliferation: It has been used to inhibit CD8 T cell proliferation and block cytotoxic function, crucial in studies of T cell biology.
• Flow Cytometry: Widely utilized as a label (with fluorochromes such as PE, APC) in flow cytometric analyses for quantifying CD8a+ T cells.
• CD8a Biology: Literature documents that CD8a associates with CD8b to form a coreceptor heterodimer on most cytotoxic T cells and interacts with MHC I for antigen recognition; some lymphocyte subsets express CD8a without CD8b.
• Cross-reactivity: Notably, 53-6.7 cross-reacts with CD8 ?-like polypeptides in some non-mouse species (e.g., Egyptian toad, Bufo regularis), supporting its evolutionary conservation and utility in comparative immunology.
• Mechanistic Insights: The truncated CD8 ?' chain, which 53-6.7 also recognizes, is thought to modulate costimulatory signaling in thymic T-cell maturation.

Experimental Recommendations and Caveats:

• Ultra-LEAF™ purified 53-6.7 is preferred for functional assays or in vivo studies due to its low endotoxin and azide-free formulation.
• Not recommended for immunohistochemistry of formalin-fixed paraffin sections due to inability to detect CD8a in this context.

In summary, clone 53-6.7 is a benchmark tool for mouse CD8+ T-cell research, frequently cited for cell depletion, functional assays, and flow cytometric analyses, and has contributed substantial mechanistic insights into T-cell biology by enabling direct manipulation or identification of CD8a-expressing cells.

Dosing regimens for clone 53-6.7—a monoclonal antibody targeting mouse CD8?—vary significantly depending on the experimental design, mouse strain, disease model, administration route, and intended immunological outcome (e.g., depletion, blocking, or staining).

In mouse models where 53-6.7 is used for depletion of CD8+ T cells, protocols typically mirror those established for similar CD8?-targeting antibodies (such as clone 2.43), with dosing regimens commonly ranging from 100??g to 500??g per mouse, administered intraperitoneally (i.p.) or intravenously (i.v.). For sustained depletion, antibodies may be given as:

• A single bolus
• Repeated doses every 3–7 days, sometimes with an initial higher dose followed by lower maintenance injections.

When clone 53-6.7 is used for blocking antigen presentation or inhibiting T cell responses, dosing can range from 10??g to 30??g per mouse per injection, particularly in therapeutic or tumor immunity studies. Lower doses are favored to minimize toxicity while maintaining efficacy, as high-dose regimens may increase risk of adverse effects (as seen with other antibodies in similar settings).

Key variables influencing regimen across models:

• Mouse strain: C57BL/6 and BALB/c are common choices; immune background may affect antibody efficacy and side-effect profile.
• Disease context: Tumor-bearing mice may show increased sensitivity to repeated antibody administration, requiring careful adjustment.
• Frequency: Dosing intervals (e.g., biweekly, weekly, or on specific days post-immunization or tumor induction) are tailored to experimental endpoints.
• Route: Most studies use intraperitoneal administration for systemic effects; intratumoral injection may be employed for local immunomodulation.

Representative regimens (from recent literature):

• 10??g to 30??g per mouse, i.p., biweekly or up to six doses for blockade or combination immunotherapy in tumor models such as MC38 or A20.
• 100??g to 500??g per mouse, i.p. or i.v., every 3–7 days for CD8+ T cell depletion in various strains and disease settings.
• Single dose vs. repeated maintenance dosing: Protocols may start with a single high dose followed by lower maintenance doses to prolong CD8+ T cell depletion or maintain immune blockade.

Additional considerations:

• The 53-6.7 clone is well-established for CD8+ T cell depletion/blockade in mouse models, but precise regimens vary in published studies. BioLegend notes its use for antigen presentation blockade and inhibition of T cell responses, but does not specify dosing, underscoring the need for protocol optimization in each experimental scenario.
• Mild to moderate hematological toxicity may occur at higher repeated doses, as demonstrated with other immune-targeting antibodies.

In summary, dosing regimens for clone 53-6.7 in mice typically range from 10??g to 500??g per mouse per injection, with administration schedules and frequency tailored to the specific mouse model, disease state, and experimental goal.