Anti-Mouse F4/80 [Clone Cl:A3-1] — Purified in vivo PLATINUM™ Functional Grade

Clone Cl:A3-1 is a rat anti-mouse F4/80 monoclonal antibody with several important in vivo applications in mouse research, primarily centered on its ability to specifically detect and interact with macrophage populations.

Imaging and Detection of Macrophages

The antibody serves as a powerful tool for visualizing macrophage infiltration in tissues. When radiolabeled with ^111^In, the anti-F4/80-A3-1 antibody specifically localizes to tissues infiltrated by macrophages and can be used to visualize tumors through microSPECT/CT imaging. In biodistribution studies, injection of 10 or 100 μg ^111^In-anti-F4/80-A3-1 resulted in substantial splenic uptake of 78 %ID/g and 31 %ID/g respectively, with tumor uptake of 1.38 %ID/g and 4.08 %ID/g at 72 hours post-injection.

Functional and Immunomodulatory Studies

Beyond imaging, clone Cl:A3-1 demonstrates functional capabilities in vivo. The antibody modulates cytokine levels released in response to Listeria monocytogenes infection, indicating its potential role in studying immune responses to pathogens. This functional activity makes it suitable for experiments examining macrophage-mediated immune processes.

Research in Disease Models

Clone Cl:A3-1 has proven particularly valuable in studying macrophage involvement in various disease contexts. The antibody can be used to identify and track tumor-associated macrophages (TAMs) in solid cancers, where macrophage presence correlates with increased metastatic potential and poor clinical outcomes. Additionally, it enables investigation of microglia (CNS macrophages) in neurological disorders such as Alzheimer's disease, multiple sclerosis, and prion diseases. In ischemic models, including retinopathy of prematurity and hind-limb ischemia, the antibody helps researchers examine macrophage participation in neovascularization processes.

The antibody is available in a low endotoxin format specifically designed for in vivo therapeutic uses and functional studies, making it particularly suitable for experiments requiring minimal immune system activation from the reagent itself.

Commonly used antibodies or proteins in combination with Cl:A3-1 (anti-mouse F4/80) for mouse macrophage identification and characterization include:

• CD11b (Integrin alpha M): Frequently paired to help distinguish macrophage subpopulations and to separate macrophages from other mononuclear phagocytes.
• CD68: Another macrophage marker often used alongside F4/80 for detailed phenotyping of mouse macrophage subsets.
• CD45: Marks all hematopoietic leukocytes, providing context for macrophage gating and identification.
• CD79b: A B cell marker, typically used for multi-color immunostaining to exclude B cells or assess tissue environments containing both cell types.
• DAPI: A nuclear stain commonly used for counterstaining in immunofluorescence or immunohistochemistry to identify nuclei.

These markers are widely used with Cl:A3-1 in both flow cytometry and tissue staining protocols for robust identification and characterization of mouse macrophages, and for distinguishing them from other immune cell types present in tissues.

Clone Cl:A3-1 is a rat monoclonal antibody extensively cited for its specificity in identifying the F4/80 antigen, a key surface marker for murine (mouse) macrophages and microglia. Key findings from its citations in scientific literature include:

• High Specificity and Versatility: Cl:A3-1 selectively binds the F4/80 antigen, enabling identification of various macrophage populations such as Kupffer cells (liver), Langerhans cells (skin), peritoneal macrophages, splenic red pulp macrophages, and importantly, microglia in the central nervous system. It is used in applications such as flow cytometry, immunohistochemistry (including on paraffin-embedded tissues), and functional in vivo studies.

• Cancer and Tumor Microenvironment Research: Clone Cl:A3-1 is widely used to label tumor-associated macrophages (TAMs) in cancer models. TAM presence (marked by F4/80 staining) has been correlated with increased metastatic potential and poor prognosis in a variety of cancers. Staining with Cl:A3-1 is a standard method for identifying these cells within tumors.

• Neuroimmunology and Microglia Studies: It is an established reagent for detecting microglia in the CNS, facilitating research into neurological diseases such as Alzheimer's, multiple sclerosis, and prion diseases.

• In Vivo Imaging & Tracing: When radiolabeled (e.g., ^111^In-anti-F4/80-A3-1), this antibody enables non-invasive imaging of macrophages, particularly in tumors, spleen, and liver. Radiolabeled Cl:A3-1 specifically accumulates in macrophage-rich tissues, confirming its utility in visualizing macrophage infiltration and tumor microenvironments.

• Functional Studies: Cl:A3-1 can modulate cytokine release (e.g., during Listeria monocytogenes infection), indicating its utility beyond labeling for functional immunological assays. It does not bind Fc receptors or exhibit cytotoxicity, minimizing non-specific effects in experiments.

• Role in T Cell Immunoregulation: There is evidence F4/80 (the antigen recognized by Cl:A3-1) plays a role in the differentiation of antigen-specific CD8+ regulatory T cells (Tregs). Studies in F4/80 (Adgre1) knockout mice indicate involvement in autoimmune regulation and Treg generation, suggesting Cl:A3-1 may be useful in studies of immune tolerance and autoimmune disease.

• Characterization of Macrophage Heterogeneity: Quantitative studies using Cl:A3-1 reveal heterogeneity among tissue macrophage populations, e.g., differential F4/80 expression between monocytes, dendritic cells, and mature macrophages.

• Reliability and Widespread Reproducibility: Hundreds of independent studies confirm consistent, reproducible results using Cl:A3-1 in mouse tissues, making it a gold standard for murine macrophage identification and functional studies.

These findings highlight Cl:A3-1’s foundational role in immunology, oncology, and neuroscience for accurate and reliable identification and study of mouse macrophages, microglia, and their functions in health and disease.

Dosing regimens for clone Cl:A3-1 (an anti-mouse F4/80 antibody) are not standardized and vary widely depending on the mouse model, specific application, and experimental design. Most commonly, Cl:A3-1 is used for labeling macrophages in flow cytometry, immunohistochemistry, and in vivo depletion or blocking studies, with recommended doses provided as guidelines but subject to adjustments.

Key Regimen Variations Across Models and Applications:

• Flow Cytometry (ex vivo or in vitro):

  • Typical protocol is to use 10 μL of the working dilution of Cl:A3-1 per 1 × 10^6 cells in 100 μL for labeling.
  • This dilution is standard whether isolating splenic macrophages, tumor-infiltrating macrophages, or other tissue-resident populations.

• Immunohistochemistry (IHC, immunofluorescence):

  • Dosing instructions are often given as volume per tissue section (e.g., 10–20 μL per section) and may require optimization depending on fixation, antigen retrieval methods, and tissue type.
  • No single consensus dose; users are advised to titrate based on signal and background levels.

• In Vivo Applications (e.g., cell depletion or blocking):

  • Published in vivo studies show a wide range, from 25–250 μg per mouse, often administered intraperitoneally or intravenously.
  • No universally accepted dose has been established; instead, researchers adjust according to the targeted macrophage population, mouse strain (e.g., C57BL/6 vs. BALB/c), route, and treatment schedule.
  • For instance, some studies deplete tissue macrophages with higher doses, while others use Cl:A3-1 primarily for cell labeling or modulation rather than depletion.

• Experimental Variables:

  • Mouse strain, age, sex, and health condition (e.g., injury, tumor, or infection models) can influence dosage requirements and outcomes.
  • Applications in injury vs. tumor vs. systemic disease models may warrant distinct dosing regimens even for the same tissue of interest.

Summary Table: Dosing/Application Guidelines

Essential Context:

• Cl:A3-1 is regarded as the gold standard for identifying mouse macrophages by targeting the F4/80 antigen.
• Researchers are highly encouraged to clarify dosing regimens in publications, as experimental variables can significantly impact effective dosing.
• Absence of standardization highlights the importance of preliminary titration and control experiments in each new model or application.

In summary: The dosing regimen for Cl:A3-1 depends on mouse model, strain, and purpose—while flow cytometry protocols are relatively consistent, in vivo dosing varies greatly and must be empirically determined for each setting.