Anti-Mouse Ly-6G [Clone 1A8] — Purified in vivo GOLD™ Functional Grade

Anti-Mouse Ly-6G [Clone 1A8] — Purified in vivo GOLD™ Functional Grade

Product No.: L280

[product_table name="All Top" skus="L280"]

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Clone
1A8
Target
Ly-6G
Formats AvailableView All
Product Type
Monoclonal Antibody
Alternate Names
Lymphocyte antigen 6 complex, locus G
Isotype
Rat IgG2a κ
Applications
CyTOF®
,
Depletion
,
FC
,
IHC FF
,
in vivo
,
PhenoCycler®
,
WB

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Data

Anti-Mouse Ly6G CyTOF™ Data
Clone 1A8 was used for neutrophil depletion in an acute zymosan-induced peritonitis model. 500µg of Clone 1A8 was dosed one day before the experiment. Zymosan was injected 3-hours prior to takedown.Clone 1A8 was used for neutrophil depletion in an acute zymosan-induced peritonitis model. 500µg of Clone 1A8 was dosed one day before the experiment. Zymosan was injected 3-hours prior to takedown.

Data generously provided by Dr. Nan Zhang Lab at Wistar Institute, Philadelphia, PA
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Antibody Details

Product Details

Reactive Species
Mouse
Host Species
Rat
Recommended Isotype Controls
Recommended Dilution Buffer
Immunogen
Mouse Ly-6G transfected EL-4J cell line
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
< 1.0 EU/mg as determined by the LAL method
Purity
≥95% 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.
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 Ly6G antibody (clone 1a8) for staining cells in flow cytometry is ≤ 0.25 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application.
Additional Applications Reported In Literature ?
CyTOF®
Depletion
IHC (Frozen)
IHC (Paraffin)
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
Ly6G antibody (clone 1A8) recognizes an epitope on mouse Ly6G. Clone 1A8 does not cross react with Ly6C.
Background
Ly6G antibody (clone 1A8) recognizes lymphocyte antigen 6 complex locus G6D (Ly6G; also called Gr-1), a 21-25 kDa glycosylphosphatidylinositol (GPI)-anchored protein1. Ly6G belongs to the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily, characterized by a Ly6/uPAR (LU) domain-containing a three-fingered structural motif stabilized by disulfide bonds2. Ly6G is expressed by murine neutrophils regardless of location and activation1,4,5. Eosinophils may also express low levels of Ly6G5. There is no human ortholog for Ly6G; however, a structurally related L76/uPAR protein, CD177 (also known as HNA-2a, NB1, or PRV-1) is expressed in human neutrophils and is implicated in neutropenia6. Although the exact function and ligand of Ly6G remain unknown, Ly6G ligation may impair neutrophil migration to sites of inflammation via a β2-integrin-dependent mechanism7.
Antigen Distribution
Ly6G is expressed by neutrophils.
PubMed
NCBI Gene Bank ID
Research Area
Immunology
.
Innate Immunity

Leinco Antibody Advisor

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The clone 1A8, specifically targeting the mouse Ly-6G marker, is widely used in several in vivo applications in mice. Here are some of the common applications:

  1. Neutrophil Depletion: Clone 1A8 is extensively used for in vivo neutrophil depletion in mice. It specifically targets Ly-6G, a marker exclusive to neutrophils, making it a crucial tool for studying the role of neutrophils in various disease models and biological processes.

  2. Immune Response Studies: By depleting neutrophils, researchers can investigate the immune response in contexts such as infectious diseases and inflammation.

  3. Cancer Research: Clone 1A8 is utilized in cancer studies to explore the role of neutrophils in cancer progression and immunotherapy.

  4. Inflammation Studies: The antibody helps in understanding the role of neutrophils in inflammatory processes, which is beneficial for studying conditions like autoimmune diseases and wound healing.

In addition to in vivo applications, clone 1A8 is also used in various in vitro techniques such as flow cytometry, immunohistochemistry, and immunofluorescence for analyzing neutrophil populations.

Commonly used antibodies or proteins alongside 1A8 (anti-Ly6G, a neutrophil marker) in the literature include those targeting cell lineage and immune subset markers, particularly in flow cytometry and immunohistochemistry studies.

Key antibodies frequently used with 1A8:

  • CD45: Pan-leukocyte marker (helps to gate all leukocytes for population analysis).
  • CD11b: Myeloid cell marker (identifies myeloid lineage cells such as monocytes, macrophages, and neutrophils).
  • F4/80: Macrophage marker (used to distinguish macrophages from neutrophils and other myeloid cells).
  • Ly6C: Monocyte subset marker (distinguishes monocytes and some T-cell subsets; used to avoid overlap with Ly6G and to separate neutrophils from monocytes).
  • RB6-8C5 (anti-Ly6G/Ly6C): Sometimes used for comparison as it recognizes both Ly6G and Ly6C and thus also depletes monocytes; helps demonstrate specificity of 1A8 for neutrophils in depletion experiments.
  • CD101, CXCR2, CXCR4: Used in combination in certain tumor models to assess neutrophil maturity.
  • Isotype controls (rat IgG2a): Crucial for background and nonspecific binding control in both in vivo and in vitro applications.

In summary, CD45, CD11b, F4/80, and Ly6C are the most commonly used antibodies with 1A8, especially in flow cytometric analysis to precisely identify and separate neutrophils from other myeloid and immune cell subsets. This panel allows researchers to:

  • Exclude or include myeloid populations,
  • Distinguish neutrophils from monocytes/macrophages,
  • Accurately quantify neutrophil depletion or presence.

RB6-8C5 is used primarily as a reference or for cross-validation of depletion efficacy because it also targets Ly6C, affecting additional cell types. Additional markers such as CD101, CXCR2, and CXCR4 are chosen for more specialized functional or maturity studies.

These combinations provide comprehensive immune profiling alongside 1A8, ensuring high specificity and accuracy in both depletion and identification studies.

Clone 1A8 is widely cited in scientific literature as a monoclonal antibody that specifically targets mouse Ly6G, enabling depletion of neutrophils for functional studies in vivo. Key findings from 1A8 citations highlight its critical role in uncovering neutrophil functions in immunity, disease models, and optimizing depletion protocols.

Essential context and key insights:

  • Specificity for Neutrophil Depletion: 1A8 recognizes Ly6G, a surface antigen uniquely expressed by neutrophils, allowing it to deplete this cell population without affecting other leukocytes—unlike antibodies such as RB6-8C5, which target Gr-1 and can impact monocytes as well.
  • Functional Impact on Immune Response: Neutrophil depletion using 1A8 has revealed their previously underappreciated importance in pathogen clearance (including Listeria monocytogenes), showing that loss of neutrophils impairs bacterial defense and modifies inflammatory responses.
  • Disease Models: In acute inflammation (e.g., zymosan-induced peritonitis), 1A8 treatment demonstrated that neutrophils are pivotal for the initial phase of response and tissue repair. Studies using 1A8 have shown that depletion dramatically worsens outcomes in conditions requiring neutrophil activity, such as bacterial sepsis, underscoring their protective role.
  • Protocol Optimization: Standard depletion regimens involve doses ranging from 100–500 μg, typically administered one day before experimental challenge; most protocols recommend adjusting for mouse strain, disease model, and duration needed, but not radically altering these parameters. Murinized variants (mouse IgG2a isotype) of 1A8 enable long-term depletion (up to four weeks), allowing studies in immune-deficient and tumor-bearing mouse models.
  • Preclinical Research Applications: 1A8 is commonly used in preclinical studies to model neutropenia, examine neutrophil-dependent pathology, and dissect their role in chronic inflammation and cancer progression.

Additional supporting details:

  • 1A8 can be used for flow cytometry and immunohistochemistry to detect or quantify neutrophils in mouse tissues in addition to depletion.
  • Its specificity minimizes confounding off-target effects when interpreting immune responses or disease outcomes.
  • The antibody is recommended for use in sterile environments and must be titrated carefully for optimal depletion or detection.

Summary Table: Clone 1A8 Key Findings

ApplicationKey FindingReference
Neutrophil depletionAllows specific removal of neutrophils, revealing their essential role in immunity
Disease modelsLoss of neutrophils increases susceptibility and mortality in infection and inflammation models
ProtocolsEffective doses 100–500 μg; murinized versions enable longer-term depletion (up to 4–6 weeks)
SpecificityTargets Ly6G, avoiding depletion of other myeloid cells (monocytes, DCs)
Additional usesEnables flow cytometric identification and quantification of neutrophils in mouse research

These findings have made clone 1A8 a standard tool in immunology for dissecting the roles of neutrophils in host defense, inflammation, and disease mechanisms.

Variability in 1A8 (Anti-Ly6G) Dosing Regimens Across Mouse Models

Summary
The dosing regimen for clone 1A8 (anti-Ly6G), used for neutrophil depletion in mice, generally follows a typical range of 100–250 µg per mouse, administered intraperitoneally (i.p.) three times per week. However, this regimen can vary depending on mouse strain, age, experimental model, and specific research objectives.

Key Variables Affecting 1A8 Dosing

Strain and Age
Efficacy of 1A8-induced neutrophil depletion can differ by mouse strain and age. For example, while 1A8 effectively depletes neutrophils in young BALB/c, FVB/N, and C57BL/6J mice, it may be less effective in older C57BL/6J mice or in certain infection models. Some studies have also shown differential depletion efficiency in bone marrow neutrophils depending on the strain.

Experimental Context
The nature of the study—acute versus chronic, infection versus tumor—can influence the optimal dose and frequency. For long-term or more durable depletion, some protocols may require higher or more frequent dosing, or even combination with secondary antibodies to achieve deeper depletion.

Dose Range and Administration Route

  • Standard dose: 100–250 µg per mouse, i.p.
  • Frequency: Typically every 2–3 days (three times per week)
  • Alternative dosing: Some protocols use a single high dose (e.g., two doses of 100 µg i.v. in PBS for acute depletion).
  • Per kg dosing: Reported as 7.5–20 mg/kg, but this is less common in the literature for clone 1A8 compared to per mouse dosing.

Specific Examples from the Literature

  • General depletion: Most studies use 100–250 µg/mouse i.p. every 2–3 days for neutrophil depletion in standard models (e.g., tumor, inflammation).
  • Acute models: A study depleted neutrophils in recipient mice with two doses of 100 µg 1A8 i.v. in 100 µL PBS.
  • Long-term depletion: In a study targeting older mice, three weekly injections of 50–100 µg were tested, suggesting that lower or less frequent dosing may be sufficient in some contexts, but efficacy must be validated.
  • Strain-specific differences: C57BL/6J mice, especially older ones, may require higher or more frequent doses, or alternative strategies (e.g., secondary antibody) to achieve significant depletion.
  • Efficacy limitations: 1A8 may be less effective at depleting immature neutrophils, and complete depletion is not always achieved, particularly in certain tumor or infection models.

Recommendations for Protocol Optimization

  • Validate depletion: Always confirm neutrophil depletion efficiency in your specific model using flow cytometry, especially when using a new strain, age group, or disease context.
  • Consider isotype controls: Include appropriate isotype controls to rule out non-specific effects.
  • Adjust for experimental needs: For acute studies, higher or more frequent dosing may be necessary; for chronic studies, consider the impact of repeated dosing on animal health and experimental outcomes.
  • Consult manufacturer guidelines: Refer to product datasheets and peer-reviewed protocols for strain- and model-specific recommendations.

Table: Typical 1A8 Dosing Regimens in Mouse Models

Model ContextDose (µg/mouse)RouteFrequencyNotes
General depletion100–250i.p.Every 2–3 daysMost common regimen
Acute depletion100 (x2)i.v.Single dayFor rapid, acute models
Long-term depletion50–100i.p.3x/weekTested in older mice
Per kg dosing7.5–20 mg/kgi.p.As neededLess commonly reported

Conclusion

While the standard 1A8 dosing regimen is 100–250 µg per mouse i.p. three times per week, significant variability exists based on mouse strain, age, experimental model, and desired depth of depletion. Researchers must tailor the regimen to their specific context and rigorously validate depletion efficiency, especially when working with non-standard strains, older mice, or complex disease models. Always consult recent literature and product guidelines to optimize the protocol for your study.

References & Citations

1. Fleming TJ, et al. (1993) J Immunol. 151(5):2399-408
2. Tsetlin VI. et al. (2015) Trends Pharmacol Sci. 36(2):109-23
3. Daley JM, et al. (2008) J Leukoc Biol. 83(1):64-70
4. Lee PY, et al. (2013) J Leukoc Biol. 94(4):585-594
5. Percopo CM, et al. (2017) J Leukoc Biol. 101(1):321-328.
6. Stroncek DF. et al. (2007) Curr Opin Hematol. 14(6):688-93
7. Wang JX, et al. (2012) Blood. 120(7):1489-1498
8. Gubin, M. et al. (2018) Cell. 175(4):1014–1030.e19 Journal Link
9. Lebratti, T.et al. (2021) eLife 10: e65762 Journal Link
10. 1. Tzetzo, S. L., Kramer, E. D., Mohammadpour, H., Kim, M., Rosario, S. R., Yu, H., Dolan, M., Oturkar, C. C., Morreale, B., Bogner, P. N., Stablewski, A., Benavides, F., Brackett, C. M., Ebos, J. M., Das, G. M., Opyrchal, M., Nemeth, M. J., Evans, S. S., & Abrams, S. I. (2024). Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression. iScience, 109187. https://doi.org/10.1016/j.isci.2024.109187
CyTOF®
Depletion
Flow Cytometry
IHC FF
in vivo Protocol
PhenoCycler®
General Western Blot Protocol

Certificate of Analysis

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