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

The clone RB6-8C5 is a monoclonal antibody used in in vivo mouse studies to deplete neutrophils by targeting the Gr-1 antigen (specifically Ly6G and, to a lesser extent, Ly6C) on the cell surface.

Context and usage details:

• Mechanism of Action: RB6-8C5 binds strongly to Ly6G (expressed on neutrophils and granulocytes) and weakly to Ly6C (also found on subsets of monocytes, dendritic cells, and lymphocytes), leading to depletion of these cell populations upon administration.
• Applications: It is widely used to study the role of neutrophils in various disease models, such as inflammation, infection, and tissue repair, by observing the effects when these cells are selectively removed from the system.
• Administration: RB6-8C5 is typically given intravenously or intraperitoneally in mice. The standard dosage ranges from 50 to 500??g per mouse, depending on factors like mouse strain, age, experimental model, and degree of desired depletion.
• Off-target Effects: While effective for neutrophil depletion, RB6-8C5 also depletes other Gr-1+ (Ly6C+) cells, which can include monocytes and some lymphocytes, potentially confounding results in studies where these populations play significant roles. This is a key consideration when interpreting data.
• Alternative Clones: For greater specificity—if depletion of only neutrophils (Ly6G+), but not Ly6C+ monocytes, is required—researchers may use antibody clone 1A8, as it does not bind Ly6C and thus spares these non-neutrophil populations.
• Functional Consequences: Depletion with RB6-8C5 has been shown to not only reduce neutrophils but also impact immune responses (such as increased TNF-alpha production), likely due to the loss of multiple Gr-1+ cell types.

Summary Table: RB6-8C5 in Mouse Studies

In summary, RB6-8C5 is a broadly used tool for neutrophil depletion in mice, but its effects on other Gr-1+ cells require careful control and experimental design consideration.

The correct storage temperature for sterile packaged clone RB6-8C5 monoclonal antibody depends on its formulation and manufacturer instructions:

• For most purified, unconjugated forms of RB6-8C5, store at –20°C. This temperature is recommended by Cell Signaling Technology for their rat monoclonal RB6-8C5, which remains stable for up to 60 months at –20°C.
• For fluorophore-conjugated forms (e.g., FITC or PE conjugates), store undiluted between 2°C and 8°C and do not freeze. This is advised by several suppliers, including BioLegend, for their FITC- and PE-conjugated RB6-8C5 products.

General guidelines for monoclonal antibodies:

• Do not aliquot unless specified, as some products may lose activity with freeze–thaw cycles.
• Always check the product datasheet for specific instructions, as distinct formulations (e.g., with added stabilizers or conjugates) may require different storage conditions.

If your sterile packaged RB6-8C5 is unconjugated and from Cell Signaling Technology, store at –20°C and do not aliquot. For conjugated antibodies (FITC, PE), keep at 2–8°C and avoid freezing. If uncertain, refer to your supplier’s protocol or datasheet for confirmation.

Commonly used antibodies or proteins with RB6-8C5 in the literature include markers for CD11b, Ly6C, Ly6G, and MHC II, as well as isotype controls such as rat IgG2b. Researchers also frequently use RB6-8C5 in combination with antibodies for CD4, CD8, B220 (CD45R), DX5 (NK cells), and sometimes with monoclonal antibodies like 131-2G and 130-6D for depletion or lineage tracking purposes.

Key proteins/antibodies used alongside RB6-8C5:

• CD11b: Marker for myeloid cells, allowing identification of granulocytic and monocytic populations.
• Ly6G and Ly6C: RB6-8C5 itself recognizes both; other antibodies like clone 1A8 are specific for Ly6G only.
• MHC II: Used as a marker to distinguish cell populations such as myeloid-derived suppressor cells (MDSCs).
• Isotype Controls (e.g., rat IgG2b): Employed to validate specificity in flow cytometry or immunohistochemistry analyses.
• 131-2G and 130-6D monoclonal antibodies: Sometimes combined with RB6-8C5 in depletion or neutralization studies of specific leukocyte subsets.
• Markers for CD4 (helper T cells), CD8 (cytotoxic T cells), B220/CD45R (B cells), DX5 (NK cells).

These combinations permit detailed phenotypic analysis or targeted depletion of specific leukocyte subsets, and help distinguish between neutrophils, monocytes, MDSCs, T cells, B cells, and NK cells in mouse models. The references above describe their use in protocols such as flow cytometry, immunohistochemistry, and depletion studies.

The clone RB6-8C5 is a widely used monoclonal antibody that targets the mouse Gr-1 antigen, which includes both Ly-6G and Ly-6C proteins. Here are the key findings from scientific literature regarding this clone:

1. Specificity and Reactivity: 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. However, some studies suggest that certain sub-populations of bone marrow cells may express both Ly-6G and Ly-6C simultaneously.

2. Applications: RB6-8C5 is extensively used for various applications, including:

   • Neutrophil Depletion: It is effective in depleting granulocytes, especially neutrophils, in vivo.
   • Flow Cytometry: Useful for identifying and distinguishing between Ly-6G and Ly-6C expressing cells in flow cytometric analyses.
   • Immunohistochemical Staining: Employed for staining in paraffin-embedded, acetone-fixed frozen sections, and zinc-fixed sections.
   • Western Blotting and Immunoprecipitation: Utilized for detecting Gr-1 in these analytical techniques.

3. Impact on Immune Studies: RB6-8C5 has been used to explore the roles of neutrophils in acquired immunity, contributing to a better understanding of their functions beyond innate immunity.

4. Interference with Other Antibodies: Clone RB6-8C5 can interfere with the binding of anti-mouse Ly-6G clone 1A8, but it can be used in conjunction with anti-mouse Ly-6C clone HK1.4.

These findings highlight the utility of the RB6-8C5 clone in studying myeloid cell populations and their roles in immune responses.