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

Clone M5/114.15.2 is most commonly used in vivo in mice to deplete or functionally block MHC class II-expressing cells, thereby modulating antigen presentation and CD4+ T cell responses.

Key in vivo applications include:

• Depletion of professional antigen-presenting cells (APCs): M5/114.15.2 targets MHC class II molecules (I-A/I-E) on dendritic cells, macrophages, and B cells, effectively depleting these APCs to study the consequences of impaired antigen presentation.
• Functional blockade of MHC class II: By blocking these molecules, the antibody interrupts CD4+ T cell activation, allowing analysis of T cell-dependent immune responses.
• Manipulation of immune responses: Used to investigate the role of MHC class II and CD4+ T cell interactions in disease models, transplantation, and autoimmunity by either depleting or blocking these interactions in vivo.

Important details:

• Epitope specificity: Clone M5/114.15.2 recognizes I-A^b, I-A^d, I-A^q, I-E^d, and I-E^k, but not I-A^f, I-A^k, or I-A^s.
• Not suitable for all strains: Only applicable to mouse strains expressing the recognized MHC class II alleles.
• Alternative uses: While its primary in vivo application is cell depletion and blockade, M5/114.15.2 is also used ex vivo and in vitro for flow cytometry, immunostaining, and functional blocking studies of T cell proliferation.

These properties make M5/114.15.2 a standard tool for dissecting the immunological roles of MHC class II molecules and APCs in living mice.

When using M5/114.15.2 to detect mouse MHC class II (I-A/I-E) in flow cytometry or immunohistochemistry, researchers commonly include other antibodies or proteins for identifying specific cell populations or studying immune activation. These co-used markers typically focus on major immune lineages and activation states.

Frequently used antibodies/proteins in combination with M5/114.15.2:

• CD11c: Dendritic cell marker, often combined with M5/114.15.2 to specifically distinguish dendritic cells within MHC II+ populations.
• CD11b: Monocyte and some dendritic cell marker; helps differentiate myeloid cells in immune organs.
• CD45: Pan-leukocyte marker to broadly identify total hematopoietic cells in tissues.
• CD3: T cell marker, commonly employed to exclude or analyze T cell subsets alongside MHC II detection.
• CD19 or B220 (CD45R): B cell markers; used to distinguish B cells within the MHC II+ cells.
• F4/80: Macrophage marker, helpful to discriminate macrophages among MHC II+ antigen-presenting cells.

Additional markers and proteins that may be included, depending on experiment:

• Activation markers: CD80, CD86, and MHC class I (H-2Kb or H-2Db).
• Viability dyes for live/dead cell discrimination.
• Isotype controls relevant to experimental setup.

This panel approach allows researchers to resolve, for example, dendritic cells (CD11c+ MHC II+), macrophages (F4/80+ CD11b+ MHC II+), B cells (CD19+ MHC II+), and T cells (CD3+ MHC II+ only on activated T cells) within mouse tissues or cultures. The exact markers included depend on the specific tissue, research question, and desired cell subset resolution.

For functional assays or blocking studies, M5/114.15.2 is sometimes used in conjunction with antibodies against CD4 (to target helper T cells), or in multi-color panels with cytokine or proliferation markers to assess downstream immune responses.

Clone M5/114.15.2 is a rat monoclonal antibody that is widely cited in scientific literature due to its specificity for mouse MHC class II molecules—specifically the I-A and I-E subregions. The key findings from citations involving this clone include:

• Broad Utility as an APC Marker: M5/114.15.2 detects a polymorphic determinant shared by several murine MHC class II alleles—I-A^b, I-A^d, I-A^q, I-E^d, and I-E^k—and is highly effective for identifying and analyzing antigen-presenting cells (APCs), including B cells, monocytes/macrophages, dendritic cells, and activated T cells in mice with certain H-2 haplotypes.
• Haplotype Specificity: This antibody does not react with I-A^f, I-A^k, or I-A^s, and thus cannot be used for all mouse strains. It does not react with NOD (H-2^g7) mice.
• Functional Applications:
  • Flow Cytometry: The antibody is extensively used for lineage analysis and quantification of MHC class II-expressing cells via flow cytometry.
  • Functional Inhibition: Clone M5/114.15.2 is reported to inhibit I-A–restricted T cell responses in some haplotypes (H-2^b, H-2^d, H-2^q, H-2^u), making it useful in mechanistic immunology studies.
  • Cell Sorting & In Vivo Depletion: It is employed in studies for labeling, sorting APCs, and, in some cases, depleting MHC class II–expressing cells to study their role in immune responses.
• Relevance in Immunology Research:
  • The clone forms a standard tool in immunophenotyping panels to distinguish various myeloid and lymphoid cell populations across numerous studies, including research on tumor immunology, infectious disease models, autoimmunity, and hematopoietic cell lineage tracing.
  • M5/114.15.2-cited studies have contributed insights into antigen presentation, immune cell activation, and the transcriptional regulation of MHC-II genes.

Summary Table: Main Features of M5/114.15.2 Citations

Clone M5/114.15.2 remains foundation to murine immunological research for defining and manipulating MHC class II–expressing cells, and is repeatedly cited in studies establishing core concepts in antigen presentation and immune cell biology.

Dosing regimens for clone M5/114.15.2 (an anti-mouse MHC class II antibody) vary widely across mouse models because they are tailored to factors such as the specific mouse strain, the experimental aim (e.g., depletion, blockade, or tracking of MHC class II–expressing cells), and the route of administration.

Key context and supporting details:

• Variation by Application and Strain: Dosing is not standardized and is commonly adjusted based on the targeted cell population, the immunological goal (depletion, blockade, tracking), the mouse strain (e.g., C57BL/6, NOD, BALB/c), and parameters like administration route and frequency.

• Typical Experimental Uses:

  • For flow cytometry, recommended doses are as low as ≤0.02–0.125 µg per test (with a test being a cell sample in 100 µL volume, typically 10^5–10^8 cells).
  • For in vivo blockade or depletion (e.g., to interfere with antigen presentation), reported regimens can range from 50–500 µg per mouse per injection, usually administered via intraperitoneal (IP) or intravenous (IV) routes, often in repeated doses—however, actual amounts and schedules must be optimized for each model and experimental endpoint. Exact regimens may differ based on strain sensitivity, cell population dynamics, and disease context.

• Empirical Titration: Manufacturers and protocol repositories strongly recommend empirical titration for each new model or assay to identify the minimal effective dose for the intended outcome with minimal off-target effects.

• Publication Practices: Reviewing specific published studies for your mouse model and intended experimental design is the standard method to guide initial dose selection, followed by laboratory titration.

In summary, there is no universal dosing regimen; dosing must be empirically determined and is influenced by experimental variables, particularly the purpose (e.g., functional blockade, depletion, tracking), mouse strain, and mode of antibody administration. For analytical applications (such as flow cytometry), doses are much lower than for in vivo functional studies such as depletion.