LY6G Antibody, Biotin

What is LY6G and why is it used as a neutrophil marker?

LY6G is a 21-25 kDa glycosylphosphatidylinositol (GPI)-anchored protein also known as the myeloid differentiation antigen Gr-1. It is expressed by the myeloid lineage in a developmentally regulated manner within the bone marrow. LY6G serves as an excellent marker for neutrophils because it is consistently expressed on both bone marrow granulocytes and peripheral neutrophils regardless of their location and activation status. While monocytes only transiently express Gr-1 during their bone marrow development, the sustained expression on neutrophils makes it a reliable identifier for this cell population in research applications. LY6G belongs to the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily, characterized by a LU domain containing a three-fingered structural motif stabilized by disulfide bonds .

What are the key differences between anti-LY6G antibody clones RB6-8C5 and 1A8?

The two primary anti-LY6G antibody clones differ significantly in their specificity:

For researchers specifically targeting neutrophils without affecting other cell populations, clone 1A8 offers significant advantages over RB6-8C5 .

What applications are LY6G antibodies commonly used for in research settings?

LY6G antibodies serve multiple critical research applications:

• Flow Cytometric Analysis: The primary application where LY6G antibodies are used to identify and quantify neutrophil populations in various tissues and blood samples. Both RB6-8C5 and 1A8 clones have been validated for flow cytometry.

• Immunohistochemistry: Used to visualize neutrophil distribution within tissue sections.

• Immunoprecipitation: Employed to isolate LY6G and associated proteins for further analysis.

• Neutrophil Depletion Studies: In vivo administration to deplete neutrophils in experimental mouse models to investigate their role in various pathological conditions.

• Functional Studies: Examination of neutrophil recruitment, migration, and activation mechanisms.

For flow cytometric analysis, anti-LY6G antibodies can be used at concentrations of ≤0.125 μg per test, where a test is defined as the amount of antibody needed to stain a cell sample in a final volume of 100 μL (cell numbers can range from 10^5 to 10^8 cells/test) .

What are the optimal storage and handling conditions for LY6G antibody, biotin?

For maximum stability and retained functionality of LY6G antibody, biotin conjugates, the following conditions should be maintained:

• Storage Temperature: Store at 2-8°C (refrigerated). Do not freeze as this may damage the antibody structure.

• Formulation: LY6G antibodies are typically formulated in phosphate buffered saline (PBS, pH 7.4) with stabilizers such as:

  • 1% BSA (bovine serum albumin)

  • 0.09% sodium azide as a preservative

• Light Exposure: Minimize exposure to light, particularly important for fluorochrome-conjugated versions.

• Handling: Avoid repeated freeze-thaw cycles and excessive agitation.

• Shipping: Should be shipped at 2-8°C to maintain stability.

When properly stored, biotin-conjugated anti-LY6G antibodies maintain their activity for the period specified by the manufacturer (typically 12-24 months from date of receipt) .

How does LY6G ligation affect neutrophil migration and what are its implications for inflammatory models?

LY6G ligation has significant effects on neutrophil migration that extend beyond simple cell depletion. Research has revealed that:

• Migration Inhibition: Even doses of anti-LY6G antibodies too low to produce sustained neutropenia can inhibit neutrophil migration to inflammatory sites. This effect occurs without altering neutrophil apoptosis, suggesting a specific mechanism affecting cell mobility.

• Chemoattractant Response: LY6G ligation abrogates neutrophil migration toward various chemoattractants, including Leukotriene B4 (LTB4), in transwell systems.

• β2-Integrin Mechanism: The migration inhibition occurs via a β2-integrin-dependent mechanism. Anti-LY6G antibody impairs surface expression of β2-integrins in LTB4-stimulated neutrophils.

• Endothelial Interactions: LY6G ligation mimics CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions.

• Model-Specific Effects: In arthritis models, low-dose anti-LY6G treatment leaves tissues free of infiltrating neutrophils. Similar effects occur in thioglycollate-stimulated peritonitis models.

These findings have important methodological implications for researchers using anti-LY6G antibodies in inflammation models, as the observed effects may result from functional alteration of neutrophil migration rather than complete neutrophil depletion. This distinction is crucial when interpreting experimental results in inflammatory disease models .

What is the molecular basis for LY6G's interaction with β2-integrins and how does this affect experimental design?

The molecular interaction between LY6G and β2-integrins represents a crucial regulatory mechanism for neutrophil function:

• Physical Association: Confocal microscopy reveals colocalization of LY6G and β2-integrins on the neutrophil surface. This close association has been confirmed by both coimmunoprecipitation studies and fluorescence lifetime imaging microscopy.

• Functional Consequences: Anti-LY6G antibody binding impairs surface expression of β2-integrins in neutrophils stimulated with chemoattractants like LTB4. This impairment functionally mimics direct CD11a blockade.

• Mechanistic Insight: The migration of β2-integrin-deficient neutrophils is no longer inhibited by anti-LY6G antibodies, confirming that the anti-migratory effect of LY6G ligation is mediated through β2-integrin-dependent mechanisms.

• Experimental Considerations: When designing experiments involving anti-LY6G antibodies, researchers should account for their effects on β2-integrin function, especially in contexts where integrin-dependent processes are being studied.

• Molecular Pathway: The GPI-anchored LY6G protein likely modulates integrin activation or clustering, affecting downstream signaling required for neutrophil adhesion and migration.

This molecular interaction explains how anti-LY6G antibodies can exert functional effects even at concentrations insufficient to deplete neutrophils completely, highlighting the need for proper controls in experiments targeting neutrophil function versus neutrophil presence .

What are the critical considerations when using different anti-LY6G antibody clones for neutrophil depletion studies?

Researchers must carefully consider several factors when designing neutrophil depletion studies:

How can researchers optimize flow cytometric analysis protocols using LY6G antibody, biotin?

Optimizing flow cytometric protocols for LY6G antibody, biotin requires attention to several technical details:

• Titration: The RB6-8C5 antibody should be carefully titrated for optimal performance, with recommended starting concentrations of ≤0.125 μg per test. Each investigator should determine their own optimal working dilution for specific applications.

• Cell Preparation: For bone marrow samples, gentle cell dissociation techniques should be used to preserve surface epitopes. For blood samples, red blood cell lysis must be performed carefully to avoid affecting neutrophil surface marker expression.

• Streptavidin Conjugate Selection: When using biotinylated anti-LY6G, selection of appropriate streptavidin-fluorochrome conjugates affects sensitivity:

  • Streptavidin-PE offers high sensitivity for detecting low expression levels

  • Streptavidin-APC provides good separation from autofluorescence

  • Multicolor panels should consider spectral overlap when selecting conjugates

• Compensation Controls: Proper single-color controls are essential when using biotin-streptavidin detection systems in multicolor panels.

• Sample Handling: Analysis should be performed promptly after staining, as prolonged storage may affect marker expression. Samples can be fixed with 1-2% paraformaldehyde for short-term storage if necessary.

• Gating Strategy: To distinguish neutrophils from monocytes that might express low levels of LY6G, additional markers such as CD11b, F4/80, and Ly6C should be included in panels.

The cell number should be determined empirically but typically ranges from 10^5 to 10^8 cells/test in a final volume of 100 μL .

What is the relationship between LY6G expression patterns and neutrophil functional states in different disease models?

LY6G expression is dynamically regulated in neutrophils under different physiological and pathological conditions:

• Developmental Regulation: In the bone marrow, LY6G expression increases with granulocyte differentiation and maturation, making it a valuable marker of neutrophil developmental stages.

• Activation States: Unlike many other neutrophil markers, LY6G expression remains relatively stable regardless of neutrophil location and activation state, providing a reliable identifier across different experimental conditions.

• Disease-Specific Patterns:

  • In inflammatory arthritis models, tissue-infiltrating neutrophils maintain LY6G expression, and anti-LY6G antibody treatment prevents this infiltration

  • In wound healing models, neutrophil accumulation at wound sites can be effectively tracked through LY6G expression

• Correlation with Function: While LY6G expression itself may not change dramatically with activation, its functional association with β2-integrins suggests it plays a regulatory role in neutrophil adhesion and migration capabilities.

• Comparative Analysis: Unlike human neutrophils, which lack a direct ortholog of LY6G but express the structurally related CD177 protein, mouse neutrophils consistently express LY6G, making it an ideal marker for tracking mouse neutrophil behavior.

This stable expression pattern makes LY6G particularly valuable for identifying neutrophils across various disease models and activation states, though researchers should be aware that eosinophils may also express low levels of LY6G, potentially requiring additional markers for complete discrimination in some contexts .

How do partial neutrophil depletion models using low-dose anti-LY6G antibodies differ from complete depletion approaches?

The distinction between partial and complete neutrophil depletion represents an important methodological consideration in inflammation research:

• Mechanistic Differences:

  • Low-dose anti-LY6G treatment primarily impairs neutrophil recruitment rather than causing cell death

  • Complete depletion protocols eliminate circulating neutrophils through antibody-dependent cellular cytotoxicity or complement-mediated mechanisms

• Experimental Observations:

  • Low doses of anti-LY6G that do not produce sustained neutropenia can still dramatically attenuate inflammatory diseases such as arthritis

  • This effect occurs primarily through blocking neutrophil migration to inflamed tissues via β2-integrin-dependent mechanisms

• Physiological Relevance:

  • Partial depletion/recruitment blockade may better model therapeutic interventions that modulate rather than eliminate neutrophil function

  • Complete depletion models the consequences of severe neutropenia

• Experimental Design Considerations:

  • Researchers should monitor both circulating neutrophil counts and tissue infiltration

  • Flow cytometric analysis of blood and tissues provides complementary information about neutrophil distribution

  • Timing of antibody administration relative to disease induction affects outcomes

• Interpretation Challenges:

  • Effects observed with low-dose anti-LY6G may reflect functional modulation rather than depletion

  • Distinguishing between absence of neutrophils and presence of functionally impaired neutrophils requires careful experimental controls

This nuanced understanding of how anti-LY6G antibodies affect neutrophil behavior at different doses provides researchers with additional experimental approaches to investigate neutrophil contributions to disease processes .

What controls should be included when using LY6G antibody, biotin in flow cytometry experiments?

Proper experimental controls are essential for accurate interpretation of LY6G staining:

• Isotype Controls:

  • For RB6-8C5 (rat IgG2b): Use rat IgG2b-biotin isotype control

  • For 1A8 (rat IgG2a): Use rat IgG2a-biotin isotype control

  • These control for non-specific binding due to Fc receptors or other interactions

• FMO Controls (Fluorescence Minus One):

  • Include all fluorochromes in your panel except the streptavidin conjugate used to detect biotinylated anti-LY6G

  • Essential for accurate gating in multicolor experiments

• Biological Controls:

  • Positive control: Bone marrow or peripheral blood samples (high neutrophil content)

  • Negative control: Lymph node samples (minimal neutrophil presence)

  • These validate antibody performance and staining protocol

• Secondary Reagent Controls:

  • Cells stained with streptavidin-fluorochrome alone (no biotinylated primary antibody)

  • Controls for non-specific binding of the streptavidin conjugate

• Blocking Controls:

  • Pre-incubation with unconjugated anti-LY6G before adding biotinylated anti-LY6G

  • Confirms epitope specificity of staining

By incorporating these controls, researchers can confidently interpret LY6G staining patterns and distinguish specific from non-specific signals .

How can researchers distinguish between LY6G and LY6C expression in complex immunological studies?

Distinguishing LY6G and LY6C expression requires careful antibody selection and multiparameter analysis:

• Antibody Clone Selection:

  • Use clone 1A8 for specific LY6G detection as it does not cross-react with LY6C

  • Use clone HK1.4 for specific LY6C detection

  • Avoid using clone RB6-8C5 alone when distinction between LY6G and LY6C is critical

• Multiparameter Analysis Strategy:

  • Include additional markers in flow cytometry panels:

    • CD11b: expressed on both neutrophils and monocytes

    • F4/80: primarily on monocytes/macrophages, minimal on neutrophils

    • CD115 (M-CSF receptor): expressed on monocytes but not neutrophils

    • Side scatter (SSC): neutrophils typically have higher granularity than monocytes

• Sequential Gating Approach:

  • First gate on CD11b+ myeloid cells

  • Then separate LY6G high (neutrophils) from LY6G low/negative cells

  • Further analyze LY6G negative population for LY6C expression to identify monocyte subsets

• Expression Pattern Analysis:

  • Neutrophils: LY6G high, LY6C intermediate

  • Inflammatory monocytes: LY6G negative, LY6C high

  • Patrolling monocytes: LY6G negative, LY6C low/intermediate

• Alternative Staining Strategy:

  • When using RB6-8C5, complement with 1A8 and HK1.4 to deconvolute Gr-1 staining patterns

This comprehensive approach allows precise identification of neutrophils, monocyte subsets, and other myeloid populations in complex samples .

How are LY6G antibodies being used to investigate neutrophil roles in emerging disease models?

LY6G antibodies have become essential tools for exploring neutrophil contributions in various disease contexts:

• Inflammatory Arthritis:

  • Anti-LY6G antibodies confirm neutrophils as key pathogenic mediators

  • Low-dose anti-LY6G treatment attenuates disease without causing complete neutropenia

  • Reveals distinct roles for neutrophil positioning versus effector functions

• Infectious Disease Models:

  • Distinguish protective versus pathological neutrophil responses

  • Identify temporal windows where neutrophil presence is beneficial or detrimental

  • Selective depletion with 1A8 provides cleaner results than RB6-8C5 by preserving monocyte populations

• Cancer Research:

  • Investigate tumor-associated neutrophil (TAN) contributions to cancer progression

  • Explore neutrophil extracellular trap (NET) formation in metastasis

  • Evaluate neutrophil-targeted therapies in combination with immunotherapies

• Sterile Inflammation:

  • Study neutrophil contribution to tissue damage following ischemia-reperfusion injury

  • Examine neutrophil recruitment dynamics in wound healing

  • Compare effects of neutrophil depletion versus migration inhibition

• Autoimmunity:

  • Define roles of neutrophils in different phases of autoimmune disease development

  • Investigate neutrophil interactions with other immune cells

  • Explore neutrophil-derived cytokines in disease perpetuation

The choice between RB6-8C5 and 1A8 clones significantly impacts experimental outcomes, with 1A8 providing more neutrophil-specific effects in most models. Understanding that LY6G ligation affects neutrophil function even without depletion has added important nuance to result interpretation .

What are the limitations and potential pitfalls when interpreting results from LY6G antibody-based experiments?

Researchers should be aware of several important caveats when using LY6G antibodies:

Understanding these limitations is essential for proper experimental design and interpretation of results. Researchers should include appropriate controls and consider complementary approaches to validate neutrophil-specific findings .