Anti-Mouse Ly-6G/Ly-6C (Gr-1) [RB6-8C5] — Purified in vivo PLATINUM™ Functional Grade

Clone RB6-8C5, an anti-mouse Ly-6G/Ly-6C (Gr-1) antibody, is commonly used in various in vivo applications in mice. Here are some of its key uses:

1. Depletion of Neutrophils and Granulocytes: RB6-8C5 is widely used to deplete neutrophils and granulocytes in mice, leading to a profound neutropenia that lasts for several days. This allows researchers to study the role of neutrophils in disease models such as inflammation, infection, and tissue repair.

2. Evaluation of Neutrophil Contributions in Disease Models: By depleting neutrophils, researchers can assess their contributions in various pathological conditions, including infections (e.g., Listeria monocytogenes, Herpes Simplex Virus type 1) and inflammatory processes.

3. Immunohistochemical Staining: The antibody is also used for staining in immunohistochemistry, allowing the visualization of Gr-1 expressing cells in paraffin-embedded or frozen tissue sections.

4. Flow Cytometry and Cell Isolation: RB6-8C5 can be used for identifying and isolating Gr-1+ cells, such as neutrophils, from mouse blood and other tissues by flow cytometry.

Commonly Used Antibodies and Proteins with RB6-8C5

RB6-8C5 is a widely used monoclonal antibody that recognizes mouse Ly-6G and Ly-6C, both referred to as Gr-1, and is mainly employed for labeling or depleting neutrophils and related myeloid populations in research involving mice. Given its broad reactivity, it is often used in combination with other antibodies and proteins to precisely identify or manipulate specific immune cell subsets.

Frequently Used Companion Antibodies and Markers

Isotype Controls

RB6-8C5 is a rat IgG2bκ isotype, so matched isotype controls (e.g., rat IgG2b) are commonly used to ensure staining specificity.

Depletion and Functional Studies

In functional studies, especially in vivo depletion experiments, RB6-8C5 is sometimes used alongside other reagents to validate specificity or to achieve more complete cell depletion. For example, combination strategies with antibodies specific for Ly6G (1A8) or other markers can help distinguish neutrophil-specific effects from broader myeloid cell alterations.

Summary Table of Typical Combinations

Key Points

• RB6-8C5 is a workhorse for labeling or depleting neutrophils and related myeloid cells in mouse models but has broad reactivity (Ly6G + Ly6C).
• CD11b (M1/70) is almost always used alongside RB6-8C5 to delineate myeloid populations.
• Ly6C (HK1.4) and Ly6G (1A8) specific antibodies are used to refine subset identification, especially when the broader reactivity of RB6-8C5 is a concern.
• MHC II, CD19, and CD3 antibodies are often included in multi-parameter panels to exclude non-myeloid cells.
• Isotype controls (rat IgG2b) are essential for validating staining specificity.

These combinations are standard in the literature for comprehensive phenotypic analysis, functional studies, and in vivo manipulation of myeloid populations in mice.

Clone RB6-8C5 is a widely cited monoclonal antibody in scientific literature, primarily used for identifying and depleting mouse neutrophils. The accumulated research has revealed several important characteristics and limitations of this antibody that researchers should consider when designing experiments.

Specificity and Target Recognition

Clone RB6-8C5 reacts predominantly with Ly-6G, a marker for mature neutrophils, but it also shows weaker reactivity with Ly-6C, which is expressed on certain lymphocytes and monocytes. The antibody recognizes the mouse Gr-1 antigen, a GPI-anchored cell surface protein bearing a single uPAR/Ly6 domain that belongs to the Ly-6 family of proteins. However, there is ongoing debate in the literature regarding the extent of cross-reactivity, with some observations suggesting that certain bone marrow cell subpopulations simultaneously express both Ly-6C and Ly-6G rather than the antibody truly cross-reacting with Ly-6C.

The antibody's broad reactivity profile means it targets Ly-6C positive subsets of monocytes, macrophages, dendritic cells, and lymphocytes in addition to neutrophils, making it challenging to draw conclusions specifically about neutrophil function alone.

Expression Patterns and Cell Identification

The Gr-1 antigen serves as a marker of myeloid differentiation, with expression levels that change during cell maturation. In the bone marrow, Gr-1 expression is low on immature myeloblasts and increases as myeloid cells mature into granulocytes. The antibody also labels macrophages and transiently stains differentiating monocytes. In peripheral contexts, RB6-8C5 specifically recognizes granulocytes.

In Vivo Depletion Capabilities and Limitations

Clone RB6-8C5 has been successfully employed for depleting mature neutrophils in vivo, achieving approximately 90% reduction of peripheral blood neutrophils. The antibody functions through the complement-dependent cytotoxicity (CDC) pathway as a rat IgG2a antibody, providing relatively fast-acting depletion.

However, critical findings have emerged regarding tissue-specific depletion efficacy. Research has clearly demonstrated that RB6-8C5 cannot eliminate hepatic myeloid-derived suppressor cells (MDSCs) and therefore should not be used to study the function of hepatic MDSC populations. Paradoxically, RB6-8C5 treatment actually enhances bone marrow MDSC migration into the liver, with the majority of transferred MDSCs accumulating in the liver of RB6-8C5-treated mice compared to controls.

Functional Impact on Target Cells

An unexpected finding is that RB6-8C5-bound MDSCs retain their immunosuppressive function. Hepatic MDSCs isolated from RB6-8C5-treated mice exhibited suppressive ability similar to non-bound hepatic MDSCs from control groups, causing dose-dependent reduction of CD8 T cell proliferation. At a 1:1 ratio, hepatic MDSCs completely blocked CD8 T cell proliferation, demonstrating that antibody binding does not neutralize the immunosuppressive capabilities of these cells.

Antibody Interference Studies

Clone RB6-8C5 impairs the binding of anti-mouse Ly-6G clone 1A8, indicating epitope overlap or steric hindrance. However, RB6-8C5 can still bind in the presence of anti-mouse Ly-6C clone HK1.4, suggesting these antibodies recognize distinct epitopes on their respective targets.

Long-Term Efficacy Concerns

A significant limitation identified in the literature is that RB6-8C5, being a rat antibody used in mice, can trigger the production of anti-rat antibodies. This immune response leads to increased clearance of the injected depletion antibodies, resulting in reduced efficacy during prolonged experiments. This has prompted the development of alternative murinized antibodies that maintain depletion efficacy while preventing anti-rat antibody production, enabling efficient neutrophil depletion for at least four weeks with consistent treatment regimens.

Overview

RB6-8C5 is a monoclonal antibody targeting the murine Ly6G/Ly6C antigens, primarily used for neutrophil (and some Gr-1⁺ monocyte) depletion in experimental mouse models. Its dosing regimens are flexible and must be optimized for each experimental context.

General Dosing Range

• Dose range: The typical dosing range for RB6-8C5 in mouse models is 50 to 500 μg per mouse per injection.
• Standard dose: Many studies use 200–250 μg per mouse injected intraperitoneally (IP) as a standard protocol.
• Dosing frequency: For sustained neutrophil depletion, injections are commonly performed every 2–3 days, though this can vary based on experimental needs.

Factors Influencing Dosing

• Mouse strain, age, and immune status: Dosing may need adjustment depending on the genetic background, age, and baseline health/immune status of the mice.
• Route of administration: Intravenous (IV) administration usually requires lower doses than intraperitoneal (IP) administration because of more direct systemic exposure with IV. IP allows for sustained but potentially less immediate exposure, sometimes necessitating higher doses for equivalent depletion.
• Degree and duration of depletion desired: Robust, rapid depletion (e.g., in acute inflammation) may require higher or more frequent dosing.
• Experimental model: The specific disease or condition being modeled (e.g., infection, inflammation, cancer) can influence the optimal dose and schedule.
• Potential off-target effects: RB6-8C5 also depletes other Gr-1⁺ (Ly6C⁺) cells, such as monocyte subsets and some dendritic cells, which may be relevant depending on the experimental question.

Practical Considerations

• Dose-response experiments: Researchers are advised to perform pilot dose-response studies to empirically determine the optimal dose for their specific model, balancing efficacy, off-target effects, and animal welfare.
• Antibody stability and cost: Higher doses increase cost and may require more frequent preparation if the antibody is unstable.
• Timing: The timing of RB6-8C5 administration relative to experimental interventions (e.g., infection, tumor challenge) can significantly impact outcomes.

Typical Dosing in Published Studies

Recommendations

• Start with 200–250 μg IP every 2–3 days as a baseline, then adjust based on pilot data and experimental requirements.
• Consider IV for acute, short-term studies where lower doses may suffice.
• Monitor depletion and off-target effects with flow cytometry or other relevant assays.
• Document strain, age, health status, and route in all reports for reproducibility.

Limitations

RB6-8C5 is not neutrophil-specific—it also affects other Ly6C⁺ populations. For neutrophil-specific depletion, the Ly6G-specific antibody 1A8 may be preferable, though it is not as widely available as RB6-8C5.

In summary, RB6-8C5 dosing in mice is highly context-dependent, with most studies using 200–250 μg IP every 2–3 days, but the optimal regimen must be empirically determined for each model, considering strain, route, depletion goals, and potential off-target effects.