Anti-Mouse CD16.2 (Clone 9E9) – Purified in vivo GOLD™ Functional Grade

Clone 9E9 is primarily used in vivo in mice to specifically block and study the function of the mouse FcγRIV receptor (CD16.2), a key activating Fc gamma receptor involved in antibody-mediated immune responses.

Common in vivo applications include:

• Blocking FcγRIV or both FcγRIV and FcγRIII: 9E9 is used to block FcγRIV function on effector myeloid cells—particularly monocytes, macrophages, neutrophils, and dendritic cells—in order to dissect its role in immune responses such as antibody-mediated cytotoxicity, inflammation, and cellular trafficking.

• Investigating effector functions of IgG2a and IgG2b: 9E9 is instrumental in clarifying the necessity of FcγRIV for mediating effector functions of mouse IgG2a and IgG2b antibodies, such as in in vivo models of platelet clearance, cytotoxicity, or autoantibody responses.

• Modulating immune complex-mediated inflammation: Blocking FcγRIV with 9E9 allows researchers to evaluate the contribution of this receptor to immune complex diseases, such as models of arthritis or systemic inflammation, where neutrophil trafficking and macrophage activation play central roles.

• Depleting or sparing specific cell types: 9E9 is used to prevent depletion of B cells or other target cells by therapeutic antibodies, thereby distinguishing which Fcγ receptor is responsible for antibody effector functions in various depletion or cytotoxicity models.

Additional applications:

• Improving specificity in immunological assays: Pre-incubation with 9E9 reduces non-antigen-specific binding by blocking FcγRIV, leading to more accurate flow cytometry or ex vivo cell functional assays.

• Functional characterization of FcγRIV: In studies of gene-targeted or transgenic mice, 9E9 is used as a pharmacological tool to complement genetic approaches, providing confirmation that observed phenotypes are due to FcγRIV activity.

Cell type specificity: FcγRIV is primarily expressed on myeloid cells (e.g., monocytes, macrophages, dendritic cells, neutrophils), but is absent on B cells, T cells, NK cells, or other lymphoid cells.

Summary: The in vivo applications of 9E9 in mice revolve around precise blockade of FcγRIV (CD16.2) to study antibody effector mechanisms, immune cell trafficking, inflammation, and therapeutic antibody function—either alone or together with other Fcγ receptors.

The antibody 9E9 is most commonly used to identify or block mouse FcγRIV (CD16.2), mainly in immunology research involving mouse models. The literature typically uses 9E9 alongside a set of other antibodies and proteins to characterize or manipulate immune cell subsets or investigate Fc receptor biology.

Commonly co-used antibodies or proteins include:

• CD16/CD32 (clone 2.4G2): Widely used as an Fc receptor blocking reagent to prevent non-specific binding in flow cytometry and functional assays. This clone blocks both FcγRII and FcγRIII, so is often combined with 9E9, which is more specific for FcγRIV, to dissect functional roles of different Fc receptors.

• Cell surface markers for immune subsets:

  • B220 (B cell marker)
  • CD3 (T cell marker)
  • CD19 (B cell marker)
  • CD11b (myeloid marker)
  • Gr1 (granulocyte marker)
  • NK1.1 (natural killer cell marker)

• Other Fc receptor antibodies:

  • Antibodies against FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16): These may be used for comparative or blocking studies to ascertain specificity and function of FcγRIV versus other Fc receptors on various immune cells.

• Isotype controls: Mouse IgG2a or irrelevant IgG to ensure specificity of staining/blocking.

• Secondary antibodies: Typically anti-IgG, with careful selection to avoid cross-reactivity, especially avoiding anti-rat IgG2b due to blocking antibody isotypes.

• Functional proteins: Fc fragments or immune complexes (ICs) are sometimes administered to trigger Fc-dependent processes, allowing for specific blockade by 9E9 versus other agents.

In summary: 9E9 (anti-CD16.2/FcγRIV) is often used with 2.4G2 (anti-CD16/CD32), other Fc receptor blockers or identifiers, classical lineage markers (B220, CD3, CD19, etc.), isotype controls, and appropriate secondaries for robust immune cell analysis and functional probing in murine immunology experiments.

The key findings from scientific literature citing clone 9E9 center on its use as a highly specific reagent to block murine FcγRIV (CD16.2), enabling researchers to elucidate the receptor’s role in immune responses, particularly those mediated by IgG2a/c antibodies.

Key findings and insights supported by the literature include:

• Specific block of FcγRIV: Clone 9E9 is widely used to specifically inhibit murine FcγRIV (CD16.2) without significant cross-reactivity to other Fc gamma receptors, which has been critical for isolating FcγRIV-mediated effects from those involving other FcγRs.

• Functional significance in immune responses: Studies using clone 9E9 demonstrate that blockade of FcγRIV:

  • Reduces IgG2a/c-mediated immune functions, including T cell activation, phagocytosis, and antibody-mediated cytotoxicity.
  • Prevents or ameliorates autoimmune and alloimmune pathologies driven by IgG2a/c, such as autoimmune hemolytic anemia, nephritis, and models of alloimmunization, making FcγRIV an attractive therapeutic target.
  • Dramatically lowers RBC alloantibody production in mouse models, indicating FcγRIV’s central role in enhancing alloimmunization.

• Experimental usage: Clone 9E9 is used in vivo and ex vivo to block FcγRIV-mediated signaling or for flow cytometric quantification of other FcγRs, helping clarify the contribution of specific FcγRs on various leukocyte subsets.

• Caveats and additional findings:

  • Blockade with 9E9 not only reduces FcγRIV expression on antigen-presenting cell subsets but can unpredictably lower FcγRII and FcγRIII levels on various cell types, highlighting the need to interpret some results with caution.
  • Despite these off-target decreases, genetic knockout studies indicate the main observed in vivo functional effects of 9E9 are indeed due to FcγRIV inhibition and not due to loss of FcγRII/III function.

• Technical relevance: 9E9 is incorporated into experimental protocols as a standard for functional blockade of FcγRIV, thereby enabling clearer dissection of receptor-specific mechanisms in antibody-driven immunity and autoimmunity.

In summary, the scientific literature establishes clone 9E9 as a foundational tool for dissecting the unique contributions of FcγRIV in the mouse immune system, with key insights into its role in antibody-mediated effector functions and disease processes.

The dosing regimens of clone 9E9, an anti-mouse FcγRIV antibody, can vary across different mouse models, primarily based on the experimental design and specific requirements of the study. Here are some general insights into how dosing regimens might differ:

1. Standard Dosage: There is limited specific information on the exact dosing regimen for clone 9E9 in mouse models. However, in one study, the anti-FcγRIV-specific antibody (clone 9E9) was administered at a dose of 10 mg/kg every four days, intraperitoneally, to CT26-bearing mice.

2. Administration Schedule: The administration schedule can vary. For example, in studies involving TIGIT blockade, the 9E9 antibody was administered one day prior to another test antibody (e.g., anti-TIGIT:mIgG2a), and dosing was continued for a total of three times.

3. Experimental Context: The dosing regimen may be adjusted based on the experimental context, such as the presence of other antibodies or the specific immune features of the mouse model being used. For instance, when studying TIGIT blockade, the interaction with FcγRIV is crucial, and dosing strategies may be optimized to ensure effective antibody function.

4. Mouse Strains and Models: While specific dosing variations based on mouse strains are not detailed, the experimental design might consider factors like receptor expression and immune response background. For example, variations in FcγR expression could influence the optimal dosing strategy.

In summary, while there is limited specific dosing information for clone 9E9 across different mouse models, the general approach involves tailoring the dosage and administration schedule to the specific experimental requirements and mouse models used.