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

Clone RB6-8C5 is widely used in vivo in mice primarily for depleting Gr-1⁺ myeloid cells, particularly to study the roles of neutrophils and, to a lesser extent, other myeloid populations in physiological and disease models.

Common in vivo applications:

• Depletion of neutrophils: RB6-8C5 is most commonly administered intravenously or intraperitoneally to induce profound and rapid neutropenia, typically lasting 3–5 days. This is used in models of inflammation, infection, tumor immunity, autoimmunity, and tissue repair to delineate the role of neutrophils by observing effects due to their absence.

• Depletion of wider Gr-1⁺ populations: RB6-8C5 also binds to Ly6C (less strongly than to Ly6G) and thus can deplete other Gr-1-expressing cells, including some monocytes, immature myeloid cells, and rarely, some lymphocyte subpopulations. This broader action must be considered when interpreting results, as monocyte and some T cell reductions are possible at certain doses or in specific experimental conditions.

• Functional assessment of myeloid cells: By depleting Gr-1⁺ cells, investigators use RB6-8C5 to assess contributions of these populations to host defense, immune regulation, tissue injury/repair, and other pathologies in vivo.

• Disease models: Commonly used in research on:

  • Infection: To evaluate how neutrophil depletion impacts control of bacterial (e.g., Listeria monocytogenes) and viral infections, and to infer roles of Gr-1⁺ cells in pathogen clearance.
  • Cancer: To understand myeloid cell involvement in tumor immunity and metastasis.
  • Inflammation: To assess neutrophil roles in acute and chronic inflammatory disease models.

Practical considerations:

• RB6-8C5 recognizes both Ly6G (high affinity) and Ly6C (lower affinity), so it is less specific for neutrophils than clone 1A8 (which is Ly6G-specific). This lack of specificity can confound interpretation, particularly in studies where monocyte involvement needs to be distinguished from neutrophil involvement.
• Effects can include not only neutropenia but also reductions in monocytes, some dendritic cells, and, in rare cases, memory CD8⁺ T cells depending on context and dose.
• For selective neutrophil depletion, clone 1A8 is now often preferred due to its exclusive Ly6G specificity, but RB6-8C5 remains standard for broader myeloid cell depletion.

Summary table: RB6-8C5 vs. 1A8

In conclusion:
RB6-8C5 is most commonly used in vivo to deplete Gr-1⁺ myeloid cells, primarily neutrophils, and to investigate the functional role of these cells in various mouse disease models, with the important caveat that it is not fully neutrophil-specific and may also deplete related cell types.

Commonly Used Antibodies and Proteins with RB6-8C5

RB6-8C5 is a monoclonal antibody widely used in immunology research, particularly for identifying and depleting mouse neutrophils, as it binds to both Ly-6G and Ly-6C (formerly known as Gr-1). Its use often involves co-staining or co-administration with other antibodies or proteins to achieve more specific cell identification, functional studies, or depletion strategies.

Antibodies Frequently Paired with RB6-8C5

• CD11b (Mac-1, Integrin alpha M)
  RB6-8C5 is commonly used in combination with anti-CD11b (clone M1/70) to identify myeloid lineage cells, especially for flow cytometric analysis of neutrophils and monocytes. CD11b is expressed on most myeloid cells, and its co-staining with RB6-8C5 helps distinguish neutrophils (Gr-1(^+) CD11b(^+)) from other populations.
• Ly-6C (clone HK1.4)
  Since RB6-8C5 binds both Ly-6G and Ly-6C, researchers sometimes use the Ly-6C-specific antibody (clone HK1.4) to discriminate Ly-6C(^{high}) monocytes from Ly-6G(^+) neutrophils. This is important because RB6-8C5 can stain both cell types, and HK1.4 can help resolve ambiguities in complex immune cell mixtures.
• 1A8 (Ly-6G-specific)
  For studies requiring exclusive identification of neutrophils, the 1A8 antibody, which is specific to Ly-6G, is often preferred over RB6-8C5. However, RB6-8C5 and 1A8 are not typically used together in the same experiment due to potential epitope masking.
• MHC II
  MHC II (major histocompatibility complex class II) antibodies are sometimes used alongside RB6-8C5 to help identify antigen-presenting cell populations, such as dendritic cells or monocytes, in complex immune cell analyses.
• CD3 and CD19
  These markers are used to identify T lymphocytes (CD3) and B lymphocytes (CD19), respectively, and are often included in multiparameter flow cytometry panels that also use RB6-8C5 for myeloid cell identification.
• Isotype Controls
  Rat IgG2b isotype controls are used to validate the specificity of RB6-8C5 staining and to set appropriate gating in flow cytometry.

Proteins and Functional Assays

• Complement-Mediated Cell Depletion
  RB6-8C5 has been reported for use in complement-mediated cell depletion assays, particularly in vivo, to specifically eliminate neutrophils for functional studies.
• Immunoprecipitation
  The antibody is also employed in immunoprecipitation experiments to isolate Ly-6G/Ly-6C expressing cells or proteins from complex mixtures.
• In Vivo Depletion
  For neutrophil depletion studies, RB6-8C5 is administered in vivo, sometimes in combination with other depletion strategies to achieve more specific or durable effects.

Typical Experimental Contexts

• Flow Cytometry
  RB6-8C5 is routinely used in multiparameter flow cytometry to identify granulocytes, often in combination with CD11b, Ly-6C, and lymphocyte markers (CD3, CD19).
• Immunohistochemistry
  The antibody is also applicable in immunohistochemistry for tissue section analysis.
• Functional Studies
  In vivo, RB6-8C5 is used to deplete neutrophils and study their role in immune responses, inflammation, and disease models.

Summary Table: Key Antibodies Used with RB6-8C5

Conclusion

RB6-8C5 is most commonly paired with CD11b for myeloid cell identification, Ly-6C (HK1.4) for monocyte discrimination, and lymphocyte markers (CD3, CD19) for comprehensive immune profiling. Isotype controls and, in some cases, MHC II antibodies are also used. For highly specific neutrophil studies, the Ly-6G-specific 1A8 antibody is preferred, but RB6-8C5 remains a staple for broad myeloid cell analysis and in vivo depletion.

Clone RB6-8C5 is a monoclonal antibody widely cited in scientific literature for its ability to identify and deplete mouse neutrophils (Ly-6G(^+)) and, to a lesser extent, Ly-6C(^+) populations, playing a critical role in studies of myeloid cell function, immune response modulation, and tumor immunology.

Key findings from RB6-8C5 citations include:

• Specificity and Reactivity

  • RB6-8C5 binds predominantly to Ly-6G, a marker for mature neutrophils, but shows weaker reactivity to Ly-6C, which is expressed on some monocytes and certain lymphocyte subsets.
  • Other studies have debated its cross-reactivity, suggesting some bone marrow subpopulations may express both Ly-6C and Ly-6G.

• Functional Use

  • The antibody is extensively used for depletion of granulocytes/neutrophils in vivo, allowing researchers to study neutrophil function in infection, inflammation, and cancer models.
  • It is not recommended for depletion of hepatic myeloid-derived suppressor cells (MDSCs), as its targeting appears less effective in that population.

• Experimental Outcomes

  • Administration of RB6-8C5 in tumor-bearing mice significantly depletes CD11b(^+) Gr-1(^+) cells (MDSCs), slows down tumor growth, and improves animal survival.
  • In infectious disease models, RB6-8C5-mediated neutrophil depletion demonstrates that neutrophil recruitment is critical for disease development, such as in Lyme arthritis.
  • Depletion is short-term: neutrophil numbers start to rebound within days after a single injection, so continuous dosing is employed for sustained depletion.
  • In combination with therapies that prime T cells, such as LRAST, RB6-8C5 can enhance anti-tumor immune responses by fostering improved IFN-γ secretion from T cells, although results may vary between experimental replicates.

• Technical Considerations

  • Epitope competition exists: RB6-8C5 inhibits binding of the Ly-6G-specific clone 1A8, but can be used together with Ly-6C-specific clone HK1.4 for dual staining.
  • It can be used in a variety of applications including flow cytometry, immunohistochemistry, immunoprecipitation, Western blotting, and cell depletion.

• Marker Utility

  • Gr-1 (recognized by RB6-8C5) is a cell surface antigen primarily used as a marker for myeloid differentiation in mice.

Summary Table: Major Attributes of RB6-8C5| Attribute | Details ||----------------------|--------------------------------------------------------------------------------------|| Main Target | Ly-6G (neutrophils), weaker for Ly-6C (monocytes/lymphocytes) || Key Application | Neutrophil/granulocyte depletion in vivo; identification in flow cytometry || Cross-reactivity | Debate exists; possible dual-expression, variable on bone marrow populations || Epitope Competition | Inhibits clone 1A8 binding; compatible with clone HK1.4 || Disease Models | Cancer (MDSC depletion), infection (Lyme arthritis, etc.) || Technical Use | Flow cytometry, depletion, immunohistochemistry, immunoprecipitation, Western blotting|

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Dose Ranges and Administration

The anti-mouse monoclonal antibody clone RB6-8C5—targeting Gr-1 (Ly-6G/Ly-6C)—is commonly used for neutrophil depletion in murine models. Dosing regimens for RB6-8C5 in mice typically fall within 200–250 µg per mouse, administered via intraperitoneal injection every 2–3 days for sustained neutrophil depletion. However, the dosage can range widely—from 50 to 500 µg per mouse—depending on the experimental model, mouse strain, age, and the desired extent and duration of neutrophil depletion.

Influencing Factors

• Route of Administration: Intravenous (IV) delivery can achieve comparable depletion at lower doses than intraperitoneal (IP) injection due to more efficient systemic distribution. In contrast, IP administration may require higher doses to achieve the same effect.
• Experimental Context: Robust neutrophil depletion in larger mice or in models of acute inflammation/infection may necessitate higher antibody doses.
• Strain and Age: Mouse strain and age can influence antibody pharmacokinetics and pharmacodynamics, necessitating empirical titration for optimal results.
• Duration of Depletion: For prolonged or repeated depletion, more frequent dosing (every 2–3 days) is recommended. Neutropenia typically lasts 3–5 days after administration.
• Off-Target Effects: RB6-8C5 can deplete not only neutrophils but also other cell populations expressing Ly-6G (e.g., dendritic cells), so careful dose titration and monitoring are important.

General Guidelines

• Standard Regimen: 200–250 µg per mouse, IP, every 2–3 days.
• Variable Regimen: 50–500 µg per mouse, adjusted for model specifics.
• Route: IV for lower, more rapid effect; IP for sustained exposure.
• Customization: Dose-response studies are strongly recommended to tailor the regimen to your experimental needs, ensuring reproducibility and minimizing side effects.

Summary Table

Key Recommendations

• Start with standard dosing (200–250 µg/mouse, IP, every 2–3 days) and adjust based on pilot experiments and empirical readouts.
• Consider route, strain, age, and inflammatory context when designing regimens.
• Always conduct dose-response studies to optimize depletion efficiency and minimize off-target effects for your specific model.

This flexibility allows RB6-8C5 to be adapted across a wide range of mouse models, but also means that rigorous optimization is essential for reliable and reproducible results.