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

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

Product No.: G153

[product_table name="All Top" skus="G153"]

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Clone
RB6-8C5
Target
Gr-1
Formats AvailableView All
Product Type
Monoclonal Antibody
Alternate Names
Ly-6G/LY-6C, Gr-1, Myeloid
Isotype
Rat IgG2b
Applications
CyTOF®
,
Depletion
,
FC
,
IHC FF
,
IHC FFPE
,
in vivo
,
IP
,
PhenoCycler®
,
WB

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Select Product Size
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Antibody Details

Product Details

Reactive Species
Mouse
Host Species
Rat
Recommended Isotype Controls
Recommended Dilution Buffer
Immunogen
Raised against granulocytes of mouse origin
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
<0.5 EU/mg as determined by the LAL method
Purity
≥98% monomer by analytical SEC
>95% by SDS Page
Formulation
This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
Product Preparation
Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Pathogen Testing
To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s Purified Functional PLATINUM™ antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Country of Origin
USA
Shipping
Next Day 2-8°C
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for this RB6-8C5 antibody for staining cells in flow cytometry is ≤ 0.06 μg per 106 cells in a volume of 100 μl or 100 μl of whole blood. Titration of the reagent is recommended for optimal performance for each application.
Additional Applications Reported In Literature ?
CyTOF®
Depletion
Clone RB6-8C5 is suitable for In vivo depletion. However, it has been reported that clone RB6-8C5 is not suitable for depletion of hepatic myeloid derived suppressor cells (MDSCs).
IHC (Frozen)
IHC (Paraffin)
IP
WB
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
Rat Anti-Mouse Granulocytes (Clone RB6-8C5) recognizes Mouse Granulocytes . This monoclonal antibody was purified using multi-step affinity chromatography methods such as Protein A or G depending on the species and isotype. The antibody was also test by PCR and IMPACT1 certified (For more information contact your Sales Rep).
Background
Gr-1 is a 21-25 kD protein. This myeloid differentiation antigen is a glycosylphosphatidylinositol-linked protein expressed on granulocytes and macrophages. Clone RB6-8C5 antibody has been shown to inhibit the binding of the clone 1A8 antibody. Clone 1A8 monoclonal antibody reacts specifically with mouse Ly6G with no reported cross-reactivity with Ly6C.
Antigen Distribution
The Gr-1 antigen is present at various levels correlated with granulocyte differentiation and maturation. The Gr-1 antigen is expressed on other myeloid populations, but not on lymphoid or erythroid cells.1,2
PubMed
Research Area
Immunology
.
Innate Immunity

Leinco Antibody Advisor

Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.

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

TargetAntibody/Protein (Common Clones & Names)PurposeNotes
CD11bM1/70 (Anti-CD11b)Myeloid cell identificationUsed with RB6-8C5 to distinguish neutrophils, monocytes, and macrophages.
Ly-6CHK1.4 (Anti-Ly6C)Monocyte/neutrophil subset delineationLy6C is transiently expressed on monocytes; HK1.4 is recommended for specific Ly6C detection because RB6-8C5 binds both Ly6G and Ly6C.
Ly-6G1A8 (Anti-Ly6G)Neutrophil-specific labeling1A8 is highly specific for Ly6G and is used when precise neutrophil identification is needed, avoiding the broader reactivity of RB6-8C5.
MHC IIVarious (e.g., M5/114.15.2)Antigen-presenting cell identificationUsed to further characterize myeloid subsets, especially in mixed populations.
CD191D3, 6D5B cell identificationUsed in multi-parameter flow cytometry to exclude B cells from myeloid analyses.
CD3145-2C11T cell identificationUsed in multi-parameter flow cytometry to exclude T cells from myeloid analyses.

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

ApplicationCommon CombinationsPurpose
Flow CytometryRB6-8C5 + CD11b + Ly6C (HK1.4) + Ly6G (1A8)Neutrophil/monocyte subset discrimination
ImmunohistochemistryRB6-8C5 + CD11bLocalize myeloid cells in tissues
In Vivo DepletionRB6-8C5 + Isotype control (rat IgG2b)Validate specificity/depletion efficacy

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

Model ContextTypical Dose (μg/mouse)RouteFrequencyNotes
General neutrophil depletion200–250IPEvery 2–3 daysMost common in literature
Acute depletion (e.g., IV)50–200IVAs neededLower doses often sufficient
Robust/chronic depletionUp to 500IPEvery 2–3 daysFor larger mice or intense depletion

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.

References & Citations

1.) Yokoyama, Hitoshi et al. J Am Soc Nephrol. 2003 Oct;14(10):2503-15. PubMed
2.) Fleming, T. J. et al. (1993) J. of Immunol. 151(5):2399
3.) Hestdal, K. et al. (1991) J. of Immunol. 147(1):22
4.) Brummer, E. et al. (1984) J. Leuko. Bio. 36:505
CyTOF®
Depletion
Flow Cytometry
IHC FF
IHC FFPE
in vivo Protocol
Immunoprecipitation Protocol
PhenoCycler®
General Western Blot Protocol

Certificate of Analysis

Disclaimer AlertProducts are for research use only. Not for use in diagnostic or therapeutic procedures.