Anti-Mouse I-A/I-E (MHC Class II) [Clone M5/114.15.2] — Purified in vivo GOLD™ Functional Grade

Clone M5/114.15.2 is a rat IgG2b monoclonal antibody used in in vivo mouse studies primarily to target and block MHC class II molecules (I-A and I-E) present on antigen-presenting cells (APCs) such as macrophages, dendritic cells, B cells, and activated T cells.

Key uses in in vivo mouse studies:

• Blocking antigen presentation: M5/114.15.2 is commonly administered to mice to block MHC class II-mediated antigen presentation, which inhibits CD4⁺ T cell activation. This is useful for studying the role of MHC-II or CD4⁺ T cells in models of autoimmunity, transplantation, immune tolerance, and other adaptive immune processes.
• Functional assays: Ultra-low endotoxin or azide-free preparations of this antibody are specifically recommended for in vivo functional blockade, minimizing potential non-specific immune activation.
• Phenotyping of immune cells: While in vitro uses include flow cytometry and immunohistochemistry to stain and identify MHC class II-positive cells, in vivo administration of the antibody is focused on functional targeting/blockade rather than phenotyping.

Mechanism:

• By binding to MHC class II on mouse APCs, M5/114.15.2 physically prevents peptide-MHC complexes from interfacing with CD4⁺ T cell receptors and CD4 co-receptors, thus blocking T cell activation and response.

Strain specificity:

• The antibody reacts with MHC class II molecules I-Aᵇ, I-Aᵈ, I-Aᑫ, I-Eᵈ, I-Eᵏ (haplotypes H-2ᵇ, H-2ᵈ, H-2ᑫ, H-2ᵖ, H-2ʳ, H-2ᵘ), but does not react with I-Aᶠ, I-Aᵏ, I-Aˢ.

Preparation and handling:

• The antibody is affinity purified, typically supplied endotoxin-low/azide-free for in vivo use, and injected via routes such as intraperitoneal or intravenous depending on experimental design.

Additional information:

• M5/114.15.2-mediated blockade is reversible and temporal; effects wane as the antibody is cleared from circulation.
• It is frequently used in combination with other immune-modulating antibodies for more complex immune manipulation models.

In summary, in in vivo mouse studies, M5/114.15.2 is used as a functional blocker of MHC class II to manipulate antigen presentation and CD4⁺ T cell responses, offering a powerful tool for dissecting adaptive immune mechanisms.

Commonly used antibodies and proteins alongside M5/114.15.2 (a rat monoclonal antibody against mouse MHC class II I-A/I-E) in the literature primarily target markers that identify or characterize the same professional antigen-presenting cells, lymphocyte populations, or their activation states. These pairings support applications such as flow cytometry, immunohistochemistry, and immune profiling.

Frequently co-used antibodies and proteins include:

• CD11c: A dendritic cell marker, often used with M5/114.15.2 to identify dendritic cell populations.
• CD11b: Expressed on macrophages, monocytes, and some dendritic cells.
• CD45: A pan-leukocyte marker, used to broadly identify leukocyte populations.
• CD3: A T cell marker, for distinguishing T cell populations.
• CD19/B220: B cell markers to identify B lymphocytes among MHC II+ antigen-presenting cells.
• F4/80: A macrophage marker, enabling separation of macrophages from other MHC II-expressing APCs.
• CD86 and CD80: Co-stimulatory molecules often upregulated on activated antigen-presenting cells.
• CD40: Another activation marker on APCs, frequently analyzed in conjunction with MHC II levels.
• Live/dead cell viability dyes: Used in flow cytometry panels for gating out dead cells during analysis.

These combinations allow for detailed characterization of immune cell subsets, especially in mouse immunology research, leveraging the specificity of M5/114.15.2 for MHC II (I-A/I-E) molecules that define antigen-presenting cells.

Additionally, when performing functional assays or immunohistochemistry, M5/114.15.2 may be used with:

• Isotype controls (e.g., rat IgG2b) to confirm specificity.
• Fluorochrome-conjugated secondary antibodies for multiplexed detection in flow cytometry or microscopy.

This panel strategy enables discrimination of cell types, maturation stages, and activation states in immunological studies, consistent with the broad applications of M5/114.15.2 outlined in standard protocols.

Clone M5/114.15.2 is a rat monoclonal antibody widely cited in scientific literature for its specificity to mouse MHC class II molecules (I-A and I-E subregions), making it a key tool in immunology for detecting, quantifying, and depleting cells expressing these molecules.

Key findings and uses from scientific literature include:

• Target Specificity: M5/114.15.2 recognizes a polymorphic determinant shared by I-A[b], I-A[d], I-A[q], I-E[d], and I-E[k] MHC class II alloantigens, but not I-A[f], I-A[k], I-A[s], or some other haplotypes. This means it labels B cells, dendritic cells, monocytes, macrophages, and activated T lymphocytes from selected mouse strains (H-2b, H-2d, H-2q, H-2p, H-2r, H-2u), but not from H-2s, H-2f, or NOD (H-2g7) mice.

• Applications in Research:

  • Widely used for flow cytometry, immunohistochemistry, immunoprecipitation, and western blot detection of mouse MHC class II-expressing cells.
  • Crucial for identifying, sorting, or depleting antigen-presenting cells (APCs) in mouse models, supporting studies on immune cell populations and their function.
  • Often used to confirm MHC class II expression on cell surfaces or tissues in various experimental contexts.

• Functional Blocking: M5/114.15.2 is reported to inhibit I-A-restricted T cell responses in some haplotypes (H-2b, H-2d, H-2q, H-2u), but not in others (H-2f, H-2k, H-2s), enabling the study of antigen presentation and T cell activation.

• Haplotype Limitations: It does not recognize all mouse strains; thus, careful selection based on haplotype is required for experimental design.

• Isotype Information: The antibody is rat IgG2b, kappa.

The consistent finding across scientific literature is that M5/114.15.2 is a foundational tool for dissecting MHC class II immunobiology in murine models and for experimental manipulation of APCs in studies of immune responses, tolerance, and autoimmunity.

Dosing regimens of the monoclonal antibody clone M5/114.15.2 vary by application, mouse model, and experimental aim, but are most commonly dictated empirically based on the context of use, such as flow cytometry, in vitro/in vivo blocking, or depletion.

For in vitro applications (e.g., flow cytometry), Thermo Fisher reports:

• Recommended use is ~0.125 µg per 100 µL sample of mouse splenocytes, generally corresponding to 10⁵ to 10⁸ cells per test.
• For blocking experiments, the dose should be titrated for optimal effect depending on cell number and activation status.

For in vivo use, published dosing regimens in the literature for M5/114.15.2 vary because they are tailored to the immune cell population targeted, mouse strain (e.g., C57BL/6, NOD, BALB/c), route of administration, and the intended immunological effect (depletion, blockade, or tracking).

Key points from reported regimens:

• Titration is essential: The optimal amount per dose and dosing frequency often need to be determined empirically, as cell number, immune status, and strain-specific responses vary.
• In vivo blocking: Reported single doses range from 100–500 µg per mouse, often administered intraperitoneally (i.p.), sometimes repeated on subsequent days; for prolonged suppression or depletion, dosing every 2–3 days over 1–2 weeks has been reported in model system protocols (though not shown in your results, this is consistent with standard laboratory usage and would be specified in the methods section of experimental papers).
• Flow cytometry/FACS sorting: Lower doses are used to stain cell-surface MHC-II, typically under 1 µg per 10⁶ cells, as per supplier recommendations.

No direct citation from your results provides exhaustive detail on in vivo dosing across all models, but general guidance universally emphasizes careful titration and pilot studies to optimize for each experimental context.

Summary Table: Typical Ranges (for reference)

Key variables affecting regimen:

• Mouse strain and immune status
• Route of administration (intravenous or intraperitoneal are most common)
• Purpose of use (acute block vs. long-term suppression)
• Assay readout (cellular, functional, or phenotypic endpoint)

For precise guidance, researchers universally recommend pilot dose-response studies to calibrate for:

• The minimum effective concentration for target engagement.
• The minimum total dose required for sustained effect without off-target toxicity.

If you require dosing regimens for specific published mouse model studies using M5/114.15.2, searching primary literature or supplier protocols (often found in supplementary methods) will yield model- and endpoint-specific guidance, as supplier datasheets do not differentiate by mouse strain.