Anti-Mouse CD115 (CSF-1R) [Clone AFS98] — Purified in vivo PLATINUM™ Functional Grade

Use of Clone AFS98 in In Vivo Mouse Studies

Clone AFS98 is a monoclonal antibody that specifically targets the mouse colony-stimulating factor 1 receptor (CSF-1R), also known as CD115 or M-CSFR, which is expressed primarily on monocytes, macrophages, dendritic cells, and osteoclasts. Below is a summary of its in vivo use in mouse experiments, based on available literature and technical sources.

Mechanism of Action

• AFS98 binds to CSF-1R (CD115), blocking the interaction between CSF-1 and its receptor, which is critical for the survival, proliferation, and differentiation of cells in the monocytic lineage.
• Administration leads to the depletion of CSF-1R+ cells, most notably tissue macrophages, in mice.
• Some evidence suggests AFS98 may cause rapid macrophage depletion, possibly via direct toxicity or antibody-dependent mechanisms, as compared to other CSF-1R-blocking antibodies.

Experimental Procedures

• Dosing and Route: Typically, mice receive an intravenous (i.v.) injection of AFS98 (often 200 ?g per dose), administered 72 and 24 hours prior to the experimental intervention to achieve robust macrophage depletion.
• Controls: Animals are usually given a control IgG antibody at the same schedule to account for non-specific effects.
• Genetically Modified Models: In studies employing cell-type-specific depletion, AFS98 may be used in mice expressing a diphtheria toxin receptor under the control of the CSF-1R promoter (e.g., MMDTR mice), allowing for selective elimination of CSF-1R+ macrophages without affecting other cell types.
• Duration of Treatment: While short-term depletion protocols are common, prolonged studies (e.g., 6 weeks of treatment) have also been performed, revealing sustained macrophage depletion and assessing systemic physiological effects such as weight gain and bone density changes.

Applications in Research

• Macrophage Depletion Studies: AFS98 is widely used to investigate the role of macrophages in disease models, inflammation, and immune responses by transiently or chronically depleting these cells in vivo.
• Disease Models: For example, AFS98 has been employed to study the contribution of macrophages to renal pathology in diabetic mice (db/db model), with extended treatment showing protective effects.
• Comparative Studies: Researchers sometimes compare AFS98 with other CSF-1R-blocking antibodies (e.g., M279) to evaluate differences in depletion kinetics and physiological consequences.

Considerations and Limitations

• Specificity: While highly effective at depleting CSF-1R+ cells, AFS98’s effects may extend beyond simple blockade, with evidence of direct toxicity contributing to rapid cell loss.
• Off-Target Effects: The high conservation of tyrosine kinase domains among receptor families means that kinase inhibitors may have unpredictable off-target effects, whereas antibody-based approaches like AFS98 offer a more direct means of targeting CSF-1R.
• Limitations: Some macrophage-dependent functions may remain unaffected even after prolonged depletion, and the impact on control animals (e.g., wild-type vs. disease models) should be carefully assessed.

Summary Table: Key Details of AFS98 Use In Vivo

Conclusion

Clone AFS98 is a critical tool for in vivo studies in mice aiming to dissect the role of CSF-1R+ macrophages and related cells. Its use involves careful dosing, timing, and control experiments, and while it is highly effective at depleting target cells, researchers must consider potential off-target effects and limitations in interpreting phenotypic outcomes.

Storage Temperature for Sterile Packaged Clone AFS98

The correct storage temperature for sterile packaged clone AFS98 antibody depends on the intended storage duration and the specific formulation (e.g., liquid vs. purified, conjugated vs. unconjugated):

• Short-term storage (up to one month): Store sterile packaged clone AFS98 at 2–8°C. This is suitable for most research use if you plan to use the product within a month.
• Long-term storage (beyond one month): Freeze a liquid aliquot in working volumes without dilution at ? –70°C to maintain stability over extended periods. Avoid repeated freeze-thaw cycles.
• Special formulations (e.g., PE-conjugated): Some conjugated forms (e.g., PE-clone AFS98) must not be frozen; store these formulations at 2–8°C and protect from prolonged exposure to light.

Always check the datasheet for your specific lot and formulation as storage instructions may vary by manufacturer and product configuration—especially if the antibody is conjugated with a fluorophore or provided in a specialized buffer.

Summary Table

Precautions:

• Aliquot prior to freezing to avoid loss of antibody stability and to minimize contamination risk.
• Avoid repeated freeze-thaw cycles to prevent loss of activity.
• Follow manufacturer’s lot-specific datasheet for any deviations or special instructions.

Frequently Used Antibodies and Proteins with AFS98 in Scientific Literature

AFS98 is a rat monoclonal antibody targeting mouse CSF1R (also known as M-CSFR or CD115), commonly used to study macrophages and tumor-associated macrophages (TAMs) in murine models. Its typical applications include flow cytometry (FC), immunohistochemistry (IHC), and immunofluorescence (IF), often in combination with other antibodies and proteins to enable detailed immunological analyses.

Common Companion Antibodies and Proteins

• F4/80: This antibody is perhaps the most frequently used alongside AFS98. F4/80 identifies murine macrophages and is routinely employed in flow cytometry, IHC, and IF to distinguish macrophage populations. For example, in a study of tumor-infiltrating leukocytes, F4/80 and AFS98 were both used to define macrophage populations in tumor models, with F4/80 marking total macrophages and AFS98 (CD115) indicating cells that express the CSF1 receptor.
• Anti-CD163: This marker is used to identify M2-type (alternatively activated) TAMs. In tumor studies, researchers have stained with anti-CD163 in conjunction with AFS98 to assess the depletion of M2 macrophages following AFS98 treatment.
• Ly6C: Frequently used in flow cytometry panels to subdivide myeloid cell populations (e.g., F4/80+ Ly6C- cells), Ly6C is another common antibody paired with AFS98 when characterizing macrophage and leukocyte subsets in murine tissues.
• CSF-1 (M-CSF): The ligand for CSF1R, used in competition ELISA to demonstrate AFS98’s inhibitory function, where soluble murine CD115-Fc is immobilized and both human and murine CSF-1 are co-incubated with increasing AFS98 concentrations to show ligand binding blockade.
• CFSE or similar cell proliferation dyes: Although not antibodies, these dyes are sometimes used with AFS98 in macrophage depletion/replacement studies to track cellular proliferation and fate in vivo.

Experimental Workflows and Combinations

• Flow Cytometry Panels: AFS98 is often included in multicolor flow cytometry panels with F4/80, Ly6C, CD163, and other myeloid markers to identify and sort macrophage subsets, especially in characterizing tumor microenvironment and inflammation models.
• Immunohistochemistry: Double staining for F4/80 (total macrophages) and CD163 (M2 macrophages) are performed alongside AFS98 treatment to evaluate changes in macrophage recruitment and phenotype in response to therapy.
• CSF-1 Challenge Experiments: In mechanistic studies, CSF-1 is used to perturb the CSF1R/CSF-1 axis, and AFS98 is applied to block this interaction, revealing the dynamics of receptor-ligand binding and downstream effects on macrophage populations.

Summary Table of Commonly Used Antibodies and Proteins

Conclusion

AFS98 is most often paired with antibodies against F4/80, CD163, and Ly6C in experimental designs aimed at dissecting macrophage biology in mice, particularly in tumor models. These combinations allow for robust identification, quantification, and phenotypic analysis of macrophage subsets, with F4/80 and CD163 being especially prominent in immunohistochemical and immunofluorescent staining. CSF-1 is also frequently used as a protein ligand in functional assays to test AFS98's blocking activity.

Key Findings from Clone AFS98 (Anti-Mouse CD115/CSF-1R) in Scientific Literature

Biological Role of CD115/CSF-1R and Clone AFS98 Specificity

• CD115 (also known as CSF-1R or c-fms) is a critical receptor for colony-stimulating factor 1 (CSF-1, M-CSF) and interleukin-34 (IL-34), expressed on monocytes, macrophages, dendritic cells, osteoclasts, and their precursors.
• Clone AFS98 specifically recognizes mouse CD115 and is used to detect or block this receptor in experimental settings.
• CD115 signaling regulates the proliferation, differentiation, survival, and cytokine production of monocytes and macrophages, and is essential for osteoclast differentiation and bone resorption.

Therapeutic Effects in Cancer Models

• Tumor-Associated Macrophage (TAM) Depletion: Administration of the anti-CD115 monoclonal antibody (mAb) AFS98 in mouse tumor models (e.g., EL-4 lymphoma, PyMT mammary carcinoma) significantly reduced the number of tumor-associated macrophages, as evidenced by decreased staining for macrophage markers F4/80 and CD163.
• M2 Macrophage Reduction: AFS98 treatment also specifically reduced the presence of M2-polarized (pro-tumorigenic) macrophages within tumors, indicated by lower CD163 staining.
• Mechanism of Action: AFS98 blocks CSF-1/CD115 binding, preventing CSF-1-mediated signaling and leading to reduced macrophage recruitment and survival within the tumor microenvironment.
• Serum CSF-1 Accumulation: AFS98 administration caused a dramatic and sustained increase in serum CSF-1 levels, as the antibody prevents CSF-1 clearance via CD115-mediated endocytosis. This effect was dose-dependent and persisted for up to 19 days post-injection at high doses (50 mg/kg).
• Therapeutic Dose Requirement: High doses of AFS98 (25–50 mg/kg) were necessary to achieve significant therapeutic effects, likely because the antibody must compete with elevated serum CSF-1 for CD115 binding.
• Limited Impact on Angiogenesis: Despite reducing TAMs, AFS98 treatment did not detectably inhibit tumor angiogenesis, as assessed by CD31 staining.

Technical Applications

• Flow Cytometry and Western Blot: Clone AFS98 is validated for flow cytometry and Western blot applications to detect mouse CD115.
• Antibody Characteristics: AFS98 is a rat IgG2a? monoclonal antibody, available in various conjugates (e.g., PE, APC) for experimental flexibility.

Summary Table: Key Effects of Clone AFS98 in Preclinical Studies

Conclusion

Clone AFS98 is a well-characterized tool for studying and manipulating the CD115/CSF-1R pathway in mice. Its primary scientific impact lies in demonstrating that blockade of CD115 can deplete tumor-associated macrophages—including pro-tumor M2 subtypes—without affecting angiogenesis, highlighting the potential and limitations of targeting this pathway in cancer immunotherapy. The clone’s utility extends to basic research applications, where it enables precise detection and functional inhibition of CD115 in murine systems.