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

Clone 1A8, which targets the mouse Ly-6G protein, is widely used in in vivo mouse studies primarily for neutrophil depletion. Here's how it is utilized:

1. Neutrophil Depletion: The 1A8 antibody clone is administered to mice to specifically deplete neutrophils. This is achieved by targeting Ly-6G, a protein expressed on the surface of granulocytes. By removing these cells, researchers can study their role in various conditions, such as infection, inflammation, and disease models.

2. Improved Long-Term Studies: A murinized version of the 1A8 antibody has been developed to reduce the production of anti-rat antibodies in treated mice, enhancing the efficacy and duration of neutrophil depletion. This modification allows for more prolonged studies without compromising the well-being of the animals.

3. Versatility in Strains: The murinized version of the 1A8 antibody, available in mouse IgG2a and IgG2c isotypes, can be used in various mouse backgrounds, making it versatile for long-term studies and preclinical models.

Overall, the 1A8 clone facilitates detailed investigations into the roles and mechanisms of neutrophils in health and disease, providing valuable insights into immune responses and potential therapeutic strategies.

Commonly Used Antibodies and Proteins with 1A8 in the Literature

1A8 is a monoclonal antibody specific for mouse Ly-6G, a surface marker predominantly expressed on neutrophils. Its primary use is for neutrophil depletion or identification in experimental models, but it is often paired with other antibodies, proteins, or reagents for more complex ex vivo and in vivo studies.

Co-Administered Antibodies

• RB6-8C5: This is the most prominent antibody compared with 1A8. Unlike 1A8, RB6-8C5 recognizes both Ly-6G and Ly-6C, thus targeting a broader population of myeloid cells, including neutrophils and some monocytes. Studies often contrast the effects of 1A8 (Ly-6G-specific) and RB6-8C5 (Ly-6G/C-specific) to parse out the unique roles of neutrophils versus other myeloid cells.
• Isotype Controls: To ensure specificity, isotype-matched control antibodies—such as InVivoMAb rat IgG2a isotype control—are used alongside 1A8 in depletion experiments.
• Secondary Antibodies: For enhanced neutrophil depletion, especially in models where 1A8 alone is insufficient, secondary antibodies (e.g., anti-rat IgG) are sometimes added to cross-link and increase opsonization and phagocytosis by macrophages.
• Depleting Antibodies for Other Cell Types: To clarify the role of neutrophils relative to other immune cells, studies may combine 1A8 with antibodies targeting monocytes (e.g., anti-CCR2) to selectively deplete monocytes without affecting neutrophils or vice versa.

Proteins and Markers for Identification and Analysis

• CD Markers: In flow cytometry, 1A8 is frequently combined with antibodies against CD45 (pan-leukocyte), CD11b (myeloid cells), F4/80 (macrophages), and Ly-6C to further delineate immune cell subsets.
• Functional Markers: For more refined phenotyping, antibodies against CD101 and CXCR2 (markers of neutrophil maturity), or CXCR4 (associated with immature or atypical populations), are used with 1A8 to distinguish neutrophil subsets, especially in tumor or inflammation models.
• Cytokines and Chemokines: Sometimes, proteins such as recombinant cytokines (e.g., G-CSF, GM-CSF) or chemokines (e.g., CXCL12) are used in tandem with 1A8 to modulate neutrophil recruitment, activation, or survival in vivo or in vitro.

In Vitro and Imaging Applications

• Flow Cytometry and Microscopy: 1A8 is used for immunofluorescence (IF) and immunohistochemistry (IHC) to visualize neutrophils, often in combination with other cell-type-specific antibodies for co-localization studies.
• Functional Assays: To study neutrophil-mediated processes (e.g., phagocytosis, NETosis, migration), 1A8 is used alongside reagents that measure these functions, such as fluorescent beads, reactive oxygen species (ROS) probes, or extracellular trap markers.

Emerging Phenotypes and Rare Exceptions

• Atypical Ly-6G+ Populations: Recent studies have identified rare populations of Ly-6G+ macrophages in certain inflammatory contexts, prompting the use of additional macrophage markers (e.g., F4/80, CD68) to distinguish these from classic neutrophils.
• Depletion Efficiency Controls: Since 1A8’s depletion efficacy can vary by mouse strain, age, and disease model, studies often include flow cytometry validation of depletion by staining for residual neutrophils with other markers (e.g., CD11b, CD45).

Summary Table: Typical Antibody/Protein Combinations with 1A8

Key Points

• 1A8 is most commonly paired with RB6-8C5 to compare neutrophil-specific versus broader myeloid effects.
• Isotype controls are essential to confirm specificity.
• Flow cytometry panels often include CD45, CD11b, and Ly-6C for comprehensive immune profiling.
• Secondary antibodies may be added to boost depletion efficiency, especially in refractory models.
• Additional markers (CD101, CXCR2, CXCR4) help distinguish neutrophil subsets based on maturity and activation state.
• Emerging evidence highlights the need to verify depletion and phenotype due to model-specific variability.

These combinations and validations are critical for robust experimental design when using 1A8 to study neutrophil biology in mice.

Clone 1A8 is a rat monoclonal antibody that specifically targets Ly6G, a marker present on murine neutrophils, and is widely cited in scientific literature for its highly specific, efficient, and long-term depletion of neutrophils in mice.

Key findings from 1A8 citations include:

• High Specificity: 1A8 targets Ly6G, allowing for selective depletion of neutrophils without affecting monocytes or other Gr-1-expressing cells, in contrast to older antibodies like Gr-1 (RB6-8C5), which deplete both neutrophils and monocytes.
• Effective Neutrophil Depletion: 1A8 produces near-complete (>90%) and long-lasting neutrophil depletion in various mouse strains for up to four weeks. The murinized version of 1A8 (mouse IgG2a) further optimizes Fc receptor binding and complement activation, enhancing efficacy and duration while avoiding anti-rat immune responses.
• Infection Models: Studies using clone 1A8 demonstrate that neutrophils are essential for survival and bacterial clearance (e.g., in Listeria monocytogenes infections). Depletion with 1A8 increased susceptibility to high-dose infection, highlighting neutrophils’ roles in defense, particularly through TNF-? production in the liver.
• Discriminating Neutrophil Functions: Research distinguishes the specific function of neutrophils during infection, showing differences between outcomes of Gr-1 and Ly6G/1A8 antibody treatments. For instance, Gr-1 antibody depletion affects multiple cell types, complicating interpretation, while 1A8 allows for precise investigation of neutrophil roles alone.
• Tumor and Immunology Studies: 1A8 is instrumental in dissecting neutrophil function in cancer models, demonstrating its utility not just in blood but in tumor tissue. It permits specific evaluation of neutrophil involvement in immune responses and tissue infiltration.

Additional context:

• The use of 1A8 has led researchers to revise earlier conclusions from studies using Gr-1. For example, some previous findings about neutrophil necessity in infections may have over-attributed effects due to broader cell depletion with Gr-1.
• Differences in depletion strategies—Ly6G/1A8 vs. Gr-1—impact interpretations of neutrophil roles in immunity, inflammation, and tissue repair.

In summary, 1A8 citations have transformed the accuracy and specificity of neutrophil depletion experiments in mice, clarifying neutrophils' non-redundant roles in infection, immunity, and disease models by selectively targeting Ly6G.

The dosing regimens of clone 1A8 (anti-Ly6G) in mouse models typically range from 100–250??g per mouse administered intraperitoneally (i.p.), repeated three times per week or every three days. This regimen is generally used for selective and efficient neutrophil depletion in a variety of mouse strains and experimental contexts, including cancer, infection, and inflammation models.

Key context and observed variations include:

• Standard dose: 100–250??g per mouse (i.p.).
• Common schedule: Every 3 days or three times per week, to maintain neutrophil depletion.
• Alternate dosing: Some studies use lower or higher doses depending on the intended duration and depth of depletion. For example, one study administered 50??g of rat 1A8 per injection three times per week in 22–25-week-old C57BL/6, BALB/c, NXG, and SCID mice, achieving over 90% neutrophil depletion for several weeks. Dose adjustments may be made based on mouse strain, age, and disease model.
• Route: Intraperitoneal administration is standard, though intravenous (i.v.) routes are sometimes used for other antibodies but are not standard for 1A8.

Experimental use adjustments:

• Immunodeficient models (e.g., SCID, NXG): Effective with the lower end of the dose range, such as 50–100??g, and still achieves long-term depletion.
• Syngeneic tumor models and inflammation models: Often use the higher end of the dose range (200–250??g), particularly if robust and sustained depletion is required.
• Comparison to other antibodies (e.g., RB6-8C5): 1A8 is often administered at up to twice the dose used for RB6-8C5 due to differences in specificity and depletion efficiency.

There is little evidence from the provided sources of substantial regimen changes beyond these factors; most published protocols recommend sticking within these parameters and adjusting primarily for the strain, disease state, or required depletion period.

In summary:

• Dosing regimens of 1A8 in mice vary mainly in the 100–250??g per mouse i.p., three times a week, with minor adjustments for specific strains or long-term versus acute studies.
• Lower doses may be sufficient for immunodeficient mice or long-term regimens, while higher doses are more common in robust syngeneic or inflammation models.
• The regimen is chosen based on the required extent and duration of neutrophil depletion.

No sources indicate major model-specific regimen shifts outside these dosing principles.