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

The RB6-8C5 clone is a rat anti-mouse Gr-1 monoclonal antibody that is widely used in in vivo mouse studies to deplete Gr-1+ myeloid cells, particularly neutrophils, and to investigate the role of these cells in immunity and disease.

Key details on its use:

• Target Cells: RB6-8C5 primarily binds strongly to Ly6G (highly expressed on neutrophils and granulocytes) and weakly to Ly6C (expressed on monocytes, some dendritic cells, and some lymphocytes). As a result, administering RB6-8C5 can deplete not only neutrophils, but also other cell populations within the Gr-1+ compartment (e.g., Ly6C+ monocytes and some dendritic cells).

• Applications:

  • In vivo neutrophil and granulocyte depletion (most common).
  • Functional studies on the role of neutrophils/Gr-1+ cells in infection, inflammation, cancer, and tissue injury.
  • Flow cytometry and immunohistochemistry for identifying Gr-1+ populations.

• Dosage and Administration:

  • Typical dose in mice: 50–500 ?g per animal, administered intraperitoneally or intravenously.
  • Intravenous dosing achieves faster depletion but may require less antibody; intraperitoneal dosing allows for more prolonged exposure.
  • Dosing and timing should be optimized for each experimental context (strain, disease model, degree of depletion desired).

• Considerations and Caveats:

  • Due to its cross-reactivity with Ly6C, RB6-8C5 is not specific only for neutrophils and will also affect other Gr-1+ cell types (like inflammatory monocytes).
  • Off-target effects may include changes in dendritic cell populations and overall immune responses; this broader depletion is linked to altered cytokine production, such as increased TNF-alpha in some models.
  • For more neutrophil-specific depletion, some studies prefer the 1A8 clone, which targets Ly6G exclusively and spares Ly6C+ cells.

• Controls:

  • Use appropriate isotype controls and validation methods to ensure that observed experimental effects are due to Gr-1+ cell depletion and not unrelated immune modulation.

In summary, RB6-8C5 is a standard tool for depleting neutrophils and other Gr-1+ cells in mice, but its lack of strict specificity for neutrophils (binding both Ly6G and Ly6C) means researchers must interpret results with awareness of potential effects on broader myeloid populations.

Based on the available literature, several other antibodies and proteins are commonly used alongside RB6-8C5 in research studies:

Complementary Neutrophil-Specific Antibodies

1A8 antibody is frequently mentioned as a companion to RB6-8C5, with a key distinction in specificity. While RB6-8C5 reacts with both Ly-6G and Ly-6C, the 1A8 antibody is specific for Ly-6G only. This makes 1A8 particularly useful for more targeted neutrophil identification and depletion studies. Importantly, clone RB6-8C5 can impair the binding of the 1A8 antibody, which is a crucial consideration when designing experimental protocols.

HK1.4 clone targeting Ly-6C is another commonly used antibody that maintains compatibility with RB6-8C5. Unlike the interference seen with 1A8, RB6-8C5 can still effectively stain cells in the presence of the anti-mouse Ly-6C clone HK1.4.

Combination Protocols

Recent research has demonstrated the effectiveness of combination approaches using multiple antibodies together. Studies have shown that using anti-Ly6G antibodies in combination with other depleting agents can achieve more durable and controlled neutrophil depletion than using RB6-8C5 alone. These "Combo" protocols have proven particularly effective in challenging situations, such as in older mice or specific disease models where single antibody approaches may be insufficient.

Control Antibodies

Normal rat IgG serves as a standard control in neutrophil depletion experiments using RB6-8C5, as RB6-8C5 is a rat IgG2B antibody. This control is essential for validating the specificity of observed effects in experimental studies.

Functional Research Applications

The literature indicates that RB6-8C5 is commonly used in conjunction with various detection and analysis methods rather than just other antibodies. These include complement-mediated cytotoxicity assays, immunoprecipitation studies, immunohistochemical staining protocols, and Western blotting applications. Additionally, BrdU (bromodeoxyuridine) is frequently used alongside RB6-8C5 in studies examining neutrophil turnover and mobilization from bone marrow to peripheral tissues.

The choice of companion antibodies or proteins typically depends on the specific research question, with 1A8 being preferred for Ly-6G-specific applications and combination protocols being utilized when more robust neutrophil depletion is required.

Clone RB6-8C5 is a widely cited monoclonal antibody in scientific literature, primarily used for identifying and depleting mouse neutrophils and studying immune cell subsets. Its key findings can be summarized as follows:

• Target and Specificity: RB6-8C5 binds with high affinity to mouse Ly-6G and to a lesser extent Ly-6C. This allows it to label both neutrophils (Ly-6G?, Ly-6C?) and some monocytes/lymphocytes (Ly-6C?, Ly-6G?).

• Experimental Applications:

  • Widely used for neutrophil depletion in vivo, enabling investigations into the roles of neutrophils in various immune responses, infections, cancer, and inflammation.
  • Employable in flow cytometry, immunohistochemistry, immunoprecipitation, and Western blotting to identify related cell populations.
  • Used to demonstrate neutrophil contributions to acquired immunity against certain pathogens.

• Interpretation and Limitations:

  • Because RB6-8C5 binds both Ly-6G and Ly-6C, it is not exclusively neutrophil-specific, and can also deplete subsets of monocytes and certain lymphocytes expressing Ly-6C. This has significant implications for interpreting depletion experiments.
  • Not recommended for depletion of hepatic myeloid-derived suppressor cells (MDSCs), as it is less effective in the liver context.
  • RB6-8C5 interferes with the binding of other Ly-6G antibodies, such as clone 1A8, but does not interfere with anti-Ly-6C clone HK1.4.

• Impact and Cautions:

  • The use of RB6-8C5 revealed that neutrophils have previously unrecognized roles in acquired immunity to intracellular pathogens.
  • Its broad reactivity (Ly-6G and Ly-6C) means that experimental outcomes may reflect effects on multiple cell types; thus, results should be interpreted carefully, and the use of more specific antibodies (e.g., clone 1A8 for Ly-6G-specific depletion) may be preferable in some studies.
  • Combining RB6-8C5-mediated depletion with other approaches has contributed to understanding immune regulation and highlighted potential therapeutic strategies.

Summary Table: RB6-8C5 Key Findings

These findings have made RB6-8C5 a powerful reagent, but its cross-reactivity remains a critical consideration for experimental design and interpretation.

RB6-8C5 dosing regimens in mouse models typically range from 50 to 500??g per mouse per injection, with the exact protocol varying based on mouse strain, age, immune status, experimental context, and route of administration. The most common regimen is 200–250??g per mouse via intraperitoneal (i.p.) injection every 2–3 days for sustained neutrophil/granulocyte depletion.

Critical details and variations across models:

• Dose Range: Standard regimens use 200–250??g i.p. per mouse, but experimental contexts vary widely, with dose ranges reported from 50??g (for smaller/younger mice or less robust depletion) up to 500??g (for larger mice, acute inflammation, cancer, or heightened neutrophil recruitment).
• Strain and Age Dependence: The required dose can differ across mouse strains (e.g., C57BL/6, Balb/c, SCID, NXG) and with the age/weight of the animal; higher doses or more frequent dosing may be used for larger animals and in strains with more robust neutrophil recruitment.
• Route of Administration: Intravenous (i.v.) injection often allows for lower doses due to direct systemic distribution, while i.p. delivery is more common but may require higher doses for comparable depletion levels.
• Schedule: For sustained neutrophil depletion, repeated injections are needed—typically every 2–3 days, or about three times per week.
• Experimental Context: In settings of acute infection, inflammation, or tumor models, higher or more frequent dosing may be required to maintain effective depletion.
• Alternative Regimens: Some studies use dose titration starting low (e.g., 50–100??g) to minimize off-target effects and then escalate as needed. Researchers frequently monitor blood neutrophil counts to adjust dosing in real time.

Additional considerations:

• Off-target effects: RB6-8C5 recognizes both Ly6G and Ly6C and can deplete some monocyte subsets and dendritic cells, particularly at higher doses.
• Experimental optimization: Dose-response pilot studies are recommended for new models or strains to balance effective neutrophil depletion with minimized off-target toxicity.
• Alternative antibodies: For more specific neutrophil depletion, anti-Ly6G clone 1A8 (100–250??g/mouse, i.p., 2–3 times/week) is sometimes preferred.

Summary:
RB6-8C5 dosing in mice is typically 200–250??g per mouse every 2–3 days (i.p.), but regimens can vary (50–500??g, with route, strain, and context influencing exact protocols), and careful titration is recommended for each model.