Anti-Mouse CD32/CD16 [Clone 2.4G2] — Purified in vivo PLATINUM™ Functional Grade

Clone 2.4G2 is widely used in immunology research, particularly in mouse models, for its ability to block Fc gamma receptors II (CD32) and III (CD16). Common in vivo applications of this antibody include:

1. Blocking Fc Receptors: The primary use of clone 2.4G2 is to prevent non-specific binding of antibodies to Fc receptors on immune cells, enhancing the specificity of staining in flow cytometry and immunofluorescence studies.

2. Studying Immune Responses: It is utilized in studies investigating antibody-mediated immune responses, preventing interference from endogenous Fc receptor activity.

3. Functional Assays: Clone 2.4G2 is used in functional assays such as bioanalytical PK and ADA assays, and those studying biological pathways affected by mouse CD16 and CD32 proteins.

4. Modulating Fc Receptor Activity: It is used to modulate Fc receptor activity in vivo, influencing processes like antigen uptake, dendritic cell activation, and other immune responses.

5. Preventing Non-Specific Binding: This antibody is used to prevent non-specific binding during flow cytometry and other staining experiments, ensuring more accurate results.

The dosing regimen typically involves a single intraperitoneal injection of 500 µg per mouse, administered 24 hours before experimental challenge.

The 2.4G2 antibody is most frequently used as an Fc receptor blocking reagent in combination with various cell surface marker antibodies for multiparameter flow cytometry and immunofluorescence applications in mouse samples. Understanding which antibodies and proteins are commonly paired with 2.4G2 helps researchers design effective immunology experiments.

Cell Surface Marker Antibodies

When using 2.4G2 for Fc receptor blockade, researchers routinely combine it with antibodies targeting specific immune cell markers. The most commonly used cell surface marker antibodies include B220, CD3, CD19, CD11b, Gr1, and NK1.1. These antibodies enable precise identification and phenotyping of different immune cell subsets while the 2.4G2 blockade minimizes non-specific staining through Fc receptor binding.

These marker antibodies target various immune cell populations. For example, B220 and CD19 identify B cells, CD3 marks T cells, CD11b labels myeloid cells, Gr1 identifies granulocytes, and NK1.1 recognizes natural killer cells. By blocking Fc receptors with 2.4G2 before staining with these markers, researchers achieve more specific and accurate immune cell characterization.

Isotype Controls

Appropriate isotype controls are standard practice when using 2.4G2. Common isotype controls include mouse IgG2a and irrelevant IgG antibodies. These controls help establish specificity baselines and distinguish true antibody binding from background or non-specific interactions in flow cytometry and immunofluorescence experiments.

Secondary Antibodies

When 2.4G2 is used in indirect detection methods, careful selection of secondary antibodies is critical. Researchers must use anti-IgG secondary antibodies while specifically avoiding anti-rat IgG2b reagents. This precaution is necessary because 2.4G2 itself is a rat IgG2b antibody, and using anti-rat IgG2b secondary antibodies would create unwanted cross-reactivity and non-specific detection.

Functional Assay Components

In functional studies examining Fc receptor biology, 2.4G2 is frequently combined with Fc fragments and complement proteins. These combinations allow researchers to validate Fc receptor blockade effectiveness and study mechanisms such as phagocytosis and antibody-dependent cell-mediated cytotoxicity.

Additional Blocking Antibodies

For comprehensive Fc receptor blockade, researchers sometimes use 2.4G2 alongside other blocking antibodies. Notably, clone 9E9 is used to block FcγRIV, which is not effectively blocked by 2.4G2 alone. This combination ensures more complete Fc receptor blocking when studying multiple receptor types simultaneously.

The key findings from citations of clone 2.4G2 in scientific literature highlight its primary role as a monoclonal antibody used to block FcγRII (CD32) and FcγRIII (CD16) receptors on mouse immune cells. Here are the main points:

• Blocking Fc Receptors: Clone 2.4G2 is widely used to prevent non-specific binding of antibodies via their Fc regions, thereby increasing the specificity of staining for target antigens in flow cytometry and immunofluorescence experiments.

• Specificity and Applications: It is specific for CD16 and CD32 but can exhibit non-specific binding to FcγRI (CD64) under certain conditions. It is employed in various studies to characterize Fc receptor expression and to analyze functions like phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC).

• Cell Types Affected: CD16 and CD32 are expressed on several immune cells, including B cells, NK cells, macrophages, granulocytes, mast cells, and dendritic cells.

• Use in Functional Studies: Clone 2.4G2 is utilized in research on NK cell function and in studies of Fc receptor-regulated processes such as ADCC and phagocytosis.

• Dosing and Administration: Common dosages include 500 µg per mouse administered intraperitoneally 24 hours before experimentation to block Fc receptors effectively.

• Limitations and Considerations: While effective for blocking FcγRII and FcγRIII, it does not block all Fc gamma receptors, and caution is needed in choosing secondary detection reagents to avoid cross-reactivity.

Dosing regimens of clone 2.4G2 (anti-mouse CD32/CD16 monoclonal antibody) are generally standardized across mouse models, with the most common protocol being a single 500 µg intraperitoneal injection administered 24 hours prior to experimental intervention.

Context and Supporting Details:

• Standard Dose and Route: The predominant regimen for acute Fcγ receptor blockade involves a single 500 µg dose injected intraperitoneally per mouse, administered one day before the experimental procedure.
• Mouse Strains: This regimen is reported for frequently used immunological strains such as C57BL/6 and BALB/c. Major mouse strains adopt the same dosing (500 µg i.p., 24 hours prior), though protocols may be titrated for unique models, chronic studies, or repeated dosing.
• Variables: While the dose and timing tend to be consistent, adjustments may be considered for:
  • Differences in mouse strain body weight or immune background.
  • Duration of desired Fcγ receptor blockade.
  • Experimental context, such as chronic vs. acute studies.

Protocol Summary Table:

• Adjustment Considerations: There are rare reports of major protocol deviations. Variations, when present, involve titration for model-specific optimization or chronic study designs.
• Other Experimental Uses: For flow cytometry, concentrations are much lower (≤1 µg per 10^6 cells in 100 µL volume), as this application focuses on blocking Fcγ receptor-mediated non-specific antibody binding rather than in vivo modulation.

Conclusion:
Most published protocols and vendor guidelines recommend a single 500 µg intraperitoneal injection 24 hours before intervention across major mouse models, with only minor, infrequently reported variations to account for experimental specifics. Adjustments should be considered if the mouse strain, immune phenotype, or experimental goals differ substantially from those of routine acute immune blockade studies.