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

Clone 9E9 is a monoclonal antibody used in in vivo mouse studies to specifically block and investigate the function of mouse CD16.2 (Fc?RIV), a receptor involved in mediating IgG2a and IgG2b effector functions.

Application in Mouse Studies:

• Blocking Fc?RIV Function: 9E9 is primarily used to block Fc?RIV in vivo, allowing researchers to assess the receptor’s contribution to immune responses. For example, blocking studies have revealed that Fc?RIV is required for IgG2a- and IgG2b-mediated platelet clearance in living mice.
• Concurrent Inhibition of Fc?RIV and Fc?RIII: In vivo, 9E9 can also block Fc?RIII, but only when it first binds Fc?RIV on the same cell. This unique property enables concurrent investigation of both Fc?RIV and Fc?RIII functions in certain myeloid populations.
• Reduces B Cell Depletion: Blocking Fc?RIV with 9E9 has been shown to reduce B-cell depletion in mouse models, highlighting its role in antibody-dependent cytotoxic responses.
• Immune Complex Binding: 9E9 interferes with the binding of immune complexes to Fc?RIV, further elucidating its involvement in immune effector mechanisms.
• Cell Populations Studied: Fc?RIV is expressed on myeloid cells such as bone marrow dendritic cells, monocytes, macrophages, neutrophils, but is absent from lymphoid cells, T cells, B cells, NK cells, and many granulocytes.

Technical Use:

• 9E9 has also been used for labeling and quantification of Fc?RIV-expressing cells by flow cytometry in bone marrow, peripheral blood, and other tissues.

Summary Table:

References used:

• ichorbio product sheet for in vivo functional use outlining blocking and cell depletion roles.
• Leinco product sheet providing details on specificity, dual blocking with Fc?RIII, and cytokine modulation.
• Flow cytometry example for distribution studies.

If you require protocols or specific published examples, please clarify which experimental application you are most interested in (e.g., platelet clearance, B cell depletion, immune complex studies, or myeloid cell profiling).

The correct storage temperature for the sterile packaged clone 9E9 monoclonal antibody is -10°C to -25°C. Upon receipt, it is recommended to aliquot and store the antibody at -20°C, and to avoid repeated freeze/thaw cycles, as this may damage the IgG and reduce product performance.

This information is specific to the clone 9E9 antibody targeting Matrix remodeling-associated protein 8 (MXRA8), as distributed by Sigma-Aldrich.

If your product is a different "clone 9E9" (for example, an anti-LALBA antibody or another target) or a different format, storage recommendations could vary and should be verified on the specific product datasheet or package insert.

Commonly used antibodies or proteins alongside 9E9 depend on the target and experimental context, as 9E9 clones have been raised against distinct antigens (notably mouse Fc?RIV/CD16.2 and human CD46). Below are the primary uses and commonly co-applied antibodies/proteins for each:

1. 9E9 Anti-Mouse Fc?RIV/CD16.2 (Armenian Hamster IgG)

• Experimental context: Used mainly in mouse immunology for blocking or detecting Fc?RIV and sometimes Fc?RIII.
• Commonly paired antibodies/proteins:
  • Anti-mouse CD16/32 (clone 2.4G2): Used to block Fc?RII/III to distinguish specific versus nonspecific FcR-mediated binding in flow cytometry or in vivo depletion/blockade studies.
  • Anti-Ly6G, anti-F4/80: Marker antibodies for neutrophils and macrophages, frequently combined in flow cytometry panels with 9E9 to characterize myeloid cells.
  • IgG isotype controls (typically Armenian hamster IgG): For specificity controls when using 9E9 in blocking or staining protocols.
  • Detection/secondary reagents: Anti-hamster IgG (for detection), especially when 9E9 is unconjugated.
  • Functional immunoglobulin subclasses: Murine IgG2a and IgG2b, as these use Fc?RIV for effector functions; 9E9 is used in experiments testing antibody-mediated clearance or depletion.

2. 9E9 Anti-Human CD46

• Experimental context: Used to detect or study human CD46 in Western blot, flow cytometry, immunohistochemistry, etc.
• Commonly paired antibodies/proteins:
  • Cell-type markers: For co-staining in flow or tissue, anti-CD3 (T cells), anti-CD19 (B cells), anti-CD56 (NK cells), etc., to identify expression patterns in specific cell subsets.
  • Other complement regulators: Antibodies against CD55 and CD59 for comparative or multiplex studies on complement regulatory proteins.
  • Loading controls for Western blot: Anti-?-actin, anti-GAPDH, or anti-tubulin to normalize CD46 expression in lysates.
  • Secondary antibodies: Typically anti-mouse IgG when detecting mouse-derived monoclonal 9E9 in assays.
  • Parallel isotype controls: To ensure specificity in immunostaining or flow cytometry.

3. Other 9E9 Usage

• Adenovirus research: In protein chemistry, 9E9 and other monoclonals such as 1D5 are used together to map fiber protein domains and for epitope mapping in virus structure/function studies.

Summary Table of Common Combinations

The most frequently co-applied antibodies will vary with application but generally follow the above pairings for major published uses of 9E9.

Key findings from the scientific literature on clone 9E9 center on its use as a highly specific blocking antibody for mouse Fc?RIV, a type of Fc gamma receptor involved in immune responses:

• Specific inhibition of Fc?RIV: Clone 9E9 has been shown to effectively block Fc?RIV function in vivo, leading to a significant reduction in surface expression of this receptor on various murine antigen-presenting cell subsets. It is routinely used in experiments to dissect Fc?RIV-dependent pathways, particularly in studies of antibody-mediated immune responses.

• Impact on immune cell function: Administration of clone 9E9 reduces the binding and activity of IgG2a/c antibodies mediated through Fc?RIV, thereby dampening processes such as red blood cell alloantibody production and T cell activation in immune complex-driven models.

• Therapeutic implications: Studies highlight that blocking Fc?RIV using clone 9E9 can ameliorate disease in models of autoimmune and alloimmune pathology (e.g., autoimmune hemolytic anemia, rheumatoid arthritis, nephritis), showing that Fc?RIV is a promising therapeutic target.

• Cross-reactivity and experimental considerations: Treatment with clone 9E9 not only reduces Fc?RIV expression but also affects the levels of Fc?RIII and Fc?RII on certain cell populations. However, genetic studies argue that the observed effects are primarily due to Fc?RIV inhibition, not secondary to changes in other receptors. In flow cytometry protocols, clone 9E9 is essential to block Fc?RIV to avoid confounding background staining due to Fc-mediated binding by other antibodies.

• Quantification and methodology: When quantifying other Fc gamma receptors (e.g., Fc?RI, Fc?RIII), using clone 9E9 to block Fc?RIV is standard to improve specificity and avoid false positives in cell surface marker analysis.

In summary, clone 9E9 is widely cited for its utility in specifically blocking murine Fc?RIV to study its function in antibody-mediated immune responses and related disease models, with recognized downstream effects on immune cell activation and therapy development.