LY6C/G Antibody, FITC

What are Ly6C and Ly6G markers and which specific cell populations do they identify in mouse models?

Ly6C and Ly6G are cell surface glycoproteins belonging to the Ly6 family that serve as important markers for identifying and distinguishing myeloid cell populations in mice. Flow cytometric analysis using these markers reveals distinct populations:

• Neutrophils: Ly6G^Hi SSC^Int

• Classical monocytes: Ly6C^+ Ly6G^- SSC^Lo (typically MHC Class II^- CD115^+)

• Nonclassical monocytes/macrophages: Ly6C^Lo-neg Ly6G^- SSC^Lo (predominantly MHC Class II^+ CD115^-)

• Eosinophils: Ly6C^Int Ly6G^- SSC^Hi (confirmed by Siglec F expression)

After exclusion of debris, doublets, nonviable cells, and lineage markers (CD4, CD8, CD19, NK1.1, CD11c^Hi), CD11b+ cells can be sub-divided into these distinct populations using a combination of Ly6C and Ly6G vs SSC markers . This approach enables precise identification of myeloid cell subsets across various tissues and experimental models.

How does flow cytometric analysis using LY6C/G antibodies compare with other myeloid cell identification strategies?

LY6C/G antibody-based flow cytometry offers several advantages compared to alternative identification approaches:

Comparison with Gr-1-based strategies:
Using a combination of anti-Ly6C and anti-Ly6G antibodies provides more refined discrimination of myeloid cell populations compared to using the Gr-1 antibody alone. While Gr-1 antibody (clone RB6-8C5) recognizes both Ly6C and Ly6G epitopes, it doesn't allow for clear differentiation between cell types with varying expression levels of these individual markers .

Comparison with F4/80-based approaches:
Traditional approaches using F4/80 for macrophage identification can be complemented by Ly6C/Ly6G staining to better differentiate between resident and inflammatory myeloid cells. Research demonstrates that a gating strategy excluding lineage markers followed by CD11b selection and Ly6C/Ly6G discrimination provides excellent resolution of myeloid populations without requiring F4/80 staining .

Direct comparison of sorted populations using either Gr-1 or Ly6C/Ly6G surface markers revealed that these identification strategies identify similar cellular subsets, but the Ly6C/Ly6G approach provides superior resolution for distinguishing between closely related populations .

How do activated versus resting states affect Ly6C/G expression on target cell populations?

Activation state significantly impacts Ly6C/G expression patterns on myeloid and lymphoid cells:

Monocyte activation changes:

• Classical monocytes (Ly6C^hi) typically downregulate Ly6C upon tissue entry and differentiation

• Inflammatory stimuli can maintain or enhance Ly6C expression on monocytes

Lymphoid cell activation:

• CD27^+ Ly6C^- γδ T cells can upregulate Ly6C upon activation, converting to CD27^+ Ly6C^+ γδ T cells

• This phenotypic change correlates with enhanced cytotoxic function and IFNγ production

• IL-27 signaling plays a critical role in maintaining the activated Ly6C^+ phenotype on γδ T cells

Experimental observations:

• IL-27 treatment increased both the frequency and mean fluorescence intensity (MFI) of Ly6C expression on CD27^+ Ly6C^- γδ T cells by approximately 3-fold

• In IL-27 receptor-deficient mice, CD27^+ Ly6C^+ γδ T cells showed reduced expression of activation markers (CD160, NKG2A, NKp46, and IFNγ)

• Conversion of CD27^+ Ly6C^- cells into CD27^+ Ly6C^+ cells is concomitant with acquisition of their cytotoxic profile

These findings highlight the importance of considering activation state when interpreting Ly6C/G expression patterns in experimental systems.

What are the considerations when using Ly6C/G antibodies for in vivo cell depletion studies?

When designing in vivo cell depletion studies using Ly6C/G antibodies, researchers should consider several critical factors:

Antibody clone selection:

• Anti-GR-1 antibody (clone RB6-8C5): Depletes both Ly6C^hi monocytes and Ly6G^+ neutrophils

• Anti-Ly6G antibody (clone 1A8): Provides selective depletion of neutrophils (~80% depletion efficiency) without affecting Ly6C^lo monocytes

Depletion timing and duration:

• Administration 24 hours prior to experimental intervention is commonly used

• Monitor depletion efficiency throughout the experiment

Validation of depletion efficiency:

• Confirm cellular depletion in both peripheral blood and target tissues

• Use flow cytometry with appropriate markers to verify specific depletion of targeted populations

Experimental controls:

• Include isotype control antibody groups (not just PBS controls)

• Consider potential non-specific effects of antibody administration

Documented outcomes in research models:

• Anti-GR-1 treatment in viral infection models resulted in significant reductions of viral RNA levels in serum (10-fold), spleen (10-100 fold), and contralateral feet (5-fold)

• Administration of anti-Ly6G neutralizing antibody selectively depleted Ly6G+ neutrophils without affecting Ly6C^lo monocytes in tumor models

Importantly, the effects may be context-dependent. Anti-GR-1 treatment did not broadly elicit an antiviral response against all viruses, suggesting specificity to certain pathogens or mechanisms .

How do Ly6C/G expression patterns change during inflammation, infection, and disease progression?

Ly6C/G expression patterns undergo dynamic changes during disease processes, providing valuable insights into disease mechanisms and progression:

During viral infections (e.g., alphavirus):

• Increased infiltration of Ly6C^+ monocytes at infection sites

• These cells appear to facilitate alphavirus infection, promoting more rapid spread into circulation

• Depletion of Ly6C/Ly6G cells reduces viral RNA in serum, spleen, and distal tissues

Research shows that Ly6C/Ly6G cell depletion reduced viral RNA levels in serum (10-fold), spleen (10-100 fold), and contralateral feet (5-fold) during alphavirus infection. This suggests that these cells contribute to viral dissemination, as their depletion slows viral spread through the body .

In cancer progression and anti-VEGF therapy:

• Anti-VEGF therapy (DC101 treatment) induces significant increases in Ly6C^lo monocytes in tumors

• Initial increase in Ly6C^lo monocytes (day 5) followed by further increases in both Ly6C^lo monocytes and neutrophils (day 12)

• Early infiltration of Ly6C^lo monocytes during anti-VEGFR2 treatment promotes subsequent recruitment of neutrophils to tumors

In T-cell development and function:

• CD27^+ Ly6C^+ γδ T cells represent a terminally differentiated subset with distinct cytotoxic properties

• IL-27 supports the cytotoxic phenotype of CD27^+ Ly6C^+ γδ T cells

• Loss of IL-27 receptor signaling reduces the frequency of Ly6C-expressing cells and alters their phenotype

These dynamic changes highlight the importance of monitoring Ly6C/G expression patterns over time in disease models.

What is the relationship between Ly6C/G expression and functional properties of myeloid cells?

Ly6C/G expression correlates with distinct functional properties in myeloid and lymphoid populations:

Monocyte functional heterogeneity:

• Ly6C^hi monocytes: Pro-inflammatory, exhibit enhanced phagocytic capacity

• Ly6C^lo monocytes: Associated with patrolling behavior, wound healing, and immunosuppressive functions in tumor contexts

Functional implications in disease:

• Viral infection: Ly6C^+ monocytes facilitate alphavirus infection at initial sites, promoting viral dissemination

• Cancer: Ly6C^lo monocytes drive immunosuppression and confer resistance to anti-VEGF therapy

• Ly6C^lo monocytes promote subsequent recruitment of neutrophils to tumors

γδ T cell functional specialization:

• CD27^+ Ly6C^+ γδ T cells show enhanced cytotoxic capacity compared to CD27^+ Ly6C^- γδ T cells

• CD27^+ Ly6C^+ γδ T cells express higher levels of cytotoxic molecules (CD160, NKG2A, NKp46, IFNγ)

• These cells demonstrate superior ability to kill cancer cells and slow tumor growth in vivo

The research provides direct evidence of these functional differences: "CD27^+ Ly6C^+ γδ T cells are more proficient for killing cancer cells than CD27^+ Ly6C^- γδ T cells and can slow tumor growth in vivo," highlighting how Ly6C expression identifies functionally distinct subsets with clinical relevance .

How can researchers distinguish between different myeloid cell subpopulations using Ly6C/G expression patterns?

Distinguishing myeloid subpopulations using Ly6C/G expression requires a systematic gating approach combined with additional markers for full characterization:

Primary identification using Ly6C/Ly6G expression:

• Neutrophils: Ly6G^Hi SSC^Int

• Eosinophils: Ly6C^Int Ly6G^- SSC^Hi

• Classical monocytes: Ly6C^+ Ly6G^- SSC^Lo

• Nonclassical monocytes/macrophages: Ly6C^Lo-neg Ly6G^- SSC^Lo

Confirmatory markers to verify subpopulation identity:

• Neutrophils: Confirmation by CD115^- MHC-II^-

• Eosinophils: Verification using Siglec F positivity

• Classical monocytes: Typically CD115^+ MHC-II^-

• Nonclassical monocytes/macrophages: Predominantly MHC-II^+ CD115^-

For more detailed characterization:

• CCR2 expression: Typically high on Ly6C^hi monocytes

• CX3CR1 expression: Elevated on Ly6C^lo monocytes

• CXCR2: Predominantly expressed on neutrophils (Ly6G+)

A recommended gating strategy includes:

• Exclusion of debris, doublets, and dead cells

• Gate out lineage-positive cells (CD4, CD8, CD19, NK1.1, CD11c^Hi)

• Select CD11b+ cells

• Use Ly6C vs Ly6G bivariate plot to identify distinct myeloid populations

This approach enables consistent identification of neutrophils, eosinophils, and monocyte/macrophage subsets across different experimental conditions.

What alternative markers can complement Ly6C/G staining for comprehensive myeloid cell characterization?

For comprehensive myeloid cell characterization, Ly6C/G staining should be complemented with additional markers to improve resolution and functional classification:

Core myeloid markers:

• CD11b: Pan-myeloid marker, essential for initial identification

• F4/80: Widely used macrophage marker with variable expression levels

• MHC-II: Indicates antigen-presenting capacity, higher on mature macrophages

• CD115 (CSF1R): Monocyte marker, particularly useful for distinguishing monocytes from granulocytes

Subset-specific markers:

• CCR2: Predominantly expressed on Ly6C^hi monocytes

• CX3CR1: Higher expression on Ly6C^lo monocytes

• CXCR2: Primarily expressed on neutrophils

• Siglec F: Reliable marker for eosinophil identification

Activation markers:

• CD44: Associated with activation status in various cell types

• CD160, NKG2A, NKp46: Providing information on cytotoxic capacity

• T-bet: Transcription factor associated with inflammatory phenotypes

Lineage exclusion markers:

• CD4, CD8: T cell markers

• CD19: B cell marker

• NK1.1: NK cell marker

• CD11c^Hi: Dendritic cell marker

The research demonstrates the value of these combinatorial approaches: CD27^+ Ly6C^+ γδ T cells display higher expression of CD160, NKG2A, NKp46, and IFNγ compared to CD27^+ Ly6C^- γδ T cells, highlighting functional differences within phenotypically similar populations .

How do Ly6C/G-expressing cells contribute to pathogen spread in viral infection models?

Ly6C/G-expressing cells play critical roles in viral infection dynamics, particularly in alphavirus infection models:

Facilitating role in early infection:

• Ly6C+ monocytes in the skin promote systemic alphavirus infection

• These cells facilitate viral infection at the initial infection site, which accelerates spread into circulation

Impact on viral dissemination:

• Depletion of Ly6C/Ly6G cells via anti-GR-1 antibody treatment results in:

  • 10-fold reduction of viral RNA levels in serum

  • 10- to 100-fold reduction in spleen

  • 5-fold reduction in contralateral feet

  • Smaller (2-fold) reductions in the ipsilateral (infection site) feet

Virus-specific effects:

Central nervous system involvement:

• Ly6C/Ly6G myeloid cells are required for rapid CHIKV spread to the brain

• This effect appears to be independent of type I IFN signaling

Mechanisms of enhanced spread:

• Depletion of Ly6C/Ly6G cells reduces viral RNA synthesis in the skin at 8 and 18 hours post-infection

• This leads to reduced infectious virus in the draining lymph node (DLN) fluid

• Microscopy confirmed that CHIKV antigen accumulation at the periphery of the DLN was significantly reduced in anti-GR-1 treated mice

These findings suggest that targeting Ly6C/Ly6G-expressing cells could be a potential therapeutic strategy to limit viral dissemination in alphavirus infections.

How does IL-27 signaling affect Ly6C+ γδ T cell development and function?

IL-27 plays a crucial role in maintaining the development, phenotype, and function of Ly6C+ γδ T cells:

Developmental relationship:

• CD27+ Ly6C- γδ T cells represent an immature state that can convert into CD27+ Ly6C+ γδ T cells

• This conversion is accompanied by acquisition of a cytotoxic profile

• IL-27 supports this developmental progression and maintenance of the Ly6C+ phenotype

Effects on Ly6C expression:

• IL-27 treatment increases both the frequency of Ly6C-expressing γδ T cells (3-fold) and the mean fluorescence intensity (MFI) of Ly6C expression

• In IL-27 receptor-deficient (Il27ra-/-) mice:

  • Reduced frequency of CD27+ Ly6C+ γδ T cells

  • Lower Ly6C expression levels on the remaining Ly6C+ cells

  • CD27+ Ly6C+ γδ T cells from these mice show reduced Ly6C expression when transferred into recipient mice

Functional implications:

• IL-27 maintains the cytotoxic phenotype of CD27+ Ly6C+ γδ T cells

• CD27+ Ly6C+ γδ T cells in Il27ra-/- mice express lower levels of cytotoxic molecules:

  • Reduced IFNγ

  • Lower CD160

  • Decreased NKG2A

  • Diminished NKp46

• IL-27 is specifically required for CD27+ Ly6C+ cell function but is dispensable for CD27+ Ly6C- cell functions

Anti-tumor activity:

• CD27+ Ly6C+ γδ T cells control cancer progression in mice, while CD27+ Ly6C- cells cannot

• In tumor models, IL-27 receptor deficiency leads to:

  • Fewer Ly6C-expressing cells in tumors

  • Altered phenotype of tumor-infiltrating CD27+ Ly6C+ γδ T cells

  • Reduced expression of cytotoxic markers on these cells

The research demonstrates conservation between mouse and human systems: gene signatures of mouse CD27+ Ly6C+/- subsets are analogous to human mature and immature γδ-T cells, indicating potential translational relevance of these findings .

What role do Ly6C^lo monocytes play in anti-VEGF cancer therapy resistance?

Ly6C^lo monocytes contribute significantly to immunosuppression and resistance to anti-VEGF cancer therapy:

Recruitment dynamics during anti-VEGF therapy:

• Anti-VEGF therapy (DC101 treatment) induces significant increases in Ly6C^lo monocytes in tumors

• Early infiltration pattern observed:

  • Initial increase in Ly6C^lo monocytes by day 5 (380 ± 50 cells/mg compared to 180 ± 40 cells/mg in controls)

  • Further increase by day 12 (700 ± 110 cells/mg compared to 300 ± 70 cells/mg in controls)

  • Later accompanied by increased neutrophil infiltration

Orchestrating immune suppression:

• Ly6C^lo monocytes drive immunosuppression in the context of anti-VEGF cancer therapy

• These cells were previously understudied compared to other myeloid populations in the tumor microenvironment

Relationship with other myeloid populations:

• Early infiltration of Ly6C^lo monocytes during anti-VEGFR2 treatment promotes subsequent recruitment of neutrophils to tumors

• In CX3CR1-deficient mice (which lack Ly6C^lo monocytes), anti-VEGF therapy resulted in significantly reduced neutrophil infiltration compared to wild-type animals

Phenotypic characteristics:

• Ly6C^lo monocytes display high levels of CX3CR1

• In contrast, Ly6C^hi monocytes express CCR2 and Ly6G+ neutrophils express CXCR2

These findings reveal an important and previously unrecognized role for Ly6C^lo monocytes in promoting immunosuppression and resistance to anti-angiogenic therapy, suggesting that targeting these cells could potentially enhance the efficacy of anti-VEGF treatment strategies in cancer.

How can researchers optimize flow cytometric analysis of Ly6C/G-expressing cells in different tissue preparations?

Optimizing flow cytometric analysis of Ly6C/G-expressing cells requires careful consideration of tissue-specific preparation methods:

General tissue preparation considerations:

• Fresh samples typically show superior staining compared to cryopreserved material

• Protein-containing buffers (2-5% BSA or FBS) help preserve epitope recognition

• Calcium chelation (EDTA) helps prevent cell clumping but may affect some epitopes

Tissue-specific optimization approaches:

• Spleen: Direct mechanical dissociation generally preserves Ly6C/G epitopes well

  • After exclusion of debris, doublets, nonviable cells, and lineage markers (CD4, CD8, CD19, NK1.1, CD11c^Hi)

  • CD11b+ cells can be sub-divided into distinct populations using Ly6C/Ly6G vs SSC markers

• Lymph nodes: May require gentle enzymatic digestion for complete cell recovery

  • For draining lymph nodes (DLN), separation of lymphatic fluid from cellular components may be necessary to assess infectious agents

• Tumor tissue: Often requires optimization of both digestion time and enzyme concentration

  • Gentler enzymatic cocktails (e.g., Liberase rather than crude collagenase) better preserve surface epitopes

Gating strategy recommendations:

• Exclude debris, doublets, and dead cells

• Gate out lineage-positive cells (CD4, CD8, CD19, NK1.1, CD11c^Hi)

• Select CD11b+ cells

• Use Ly6C vs Ly6G bivariate plot to identify distinct myeloid populations :

  • Neutrophils: Ly6G^Hi SSC^Int

  • Eosinophils: Ly6C^Int Ly6G^- SSC^Hi (confirm with Siglec F)

  • Classical monocytes: Ly6C^+ Ly6G^- SSC^Lo

  • Nonclassical monocytes/macrophages: Ly6C^Lo-neg Ly6G^- SSC^Lo

Research demonstrates successful application of this approach across multiple tissues (spleen, lymph nodes, lung, and tumors), indicating that with proper optimization, these markers can be reliably detected in diverse tissue environments .