Anti-Mouse Ly-6C (Clone HK1.4) – Purified in vivo GOLD™ Functional Grade

Common In Vivo Applications of Clone HK1.4 in Mice

Clone Identification and Phenotyping
The rat monoclonal antibody clone HK1.4 is primarily used to identify and phenotype cells expressing Ly-6C in mice, particularly in studies focused on immunology and hematopoiesis. Ly-6C is a glycosylphosphatidylinositol (GPI)-linked surface protein expressed on various mouse immune cell populations, including monocytes, macrophages, endothelial cells, neutrophils, and some T cell subsets. By targeting Ly-6C, HK1.4 enables researchers to distinguish between different subsets of myeloid cells, which is crucial for understanding immune responses, inflammation, and immune cell trafficking.

Cell Depletion and Functional Studies
While clone HK1.4 is not primarily used for cell depletion (unlike some other Ly-6C/Ly-6G antibodies such as RB6-8C5, which targets both Ly-6C and Ly-6G for neutrophil/granulocyte depletion), it is essential for precise identification of Ly-6C-expressing cells. This specificity makes HK1.4 valuable for studies where the goal is to track, quantify, or isolate specific immune cell populations in vivo without causing depletion.

Advanced Imaging and Multiplexing
HK1.4 has been validated for use in advanced imaging platforms such as CODEX® (CO-Detection by indEXing) on fresh frozen mouse spleen tissue, enabling highly multiplexed tissue imaging and spatial analysis of immune cell populations. This application is increasingly important for detailed mapping of immune cell interactions within tissues.

In Vitro Functional Assays
In addition to in vivo applications, HK1.4 can be used in vitro to stimulate proliferation and cytokine release from certain immune cells, although this is not its primary use in murine models.

Summary Table: Key Applications of HK1.4 In Vivo

Important Notes

• Specificity: HK1.4 specifically targets Ly-6C and does not cross-react with Ly-6G, allowing clear discrimination between different myeloid subsets.
• Not for Depletion: Unlike some other Ly-6C/Ly-6G antibodies, HK1.4 is not typically used for in vivo cell depletion.
• Validation: The antibody has been rigorously tested for flow cytometry, immunohistochemistry on frozen sections, and multiplexed imaging, ensuring broad applicability in immunology research.

Conclusion

Clone HK1.4 is a cornerstone reagent for in vivo mouse studies requiring precise identification and characterization of Ly-6C-expressing cells, with applications spanning basic immunology, advanced tissue imaging, and functional immune assays. Its specificity and validation across multiple platforms make it a reliable choice for researchers investigating the role of Ly-6C+ cells in health and disease.

The most commonly co-used antibodies or proteins with HK1.4 (anti-Ly6C) in the literature are markers that help delineate immune cell subsets, particularly for monocyte phenotyping and leukocyte subset analysis.

Key co-used antibodies include:

• CD11b (Mac-1): Often combined with Ly6C to distinguish monocyte subsets (e.g., Ly6C^hiCD11b^+ for inflammatory/classical monocytes).
• CD45: Pan-leukocyte marker, useful for gating all white blood cells in flow cytometry.
• CD4 and CD8: T cell subset markers; Ly6C expression on these can vary by mouse strain.
• CD3: General T lymphocyte marker.
• F4/80: Macrophage marker, assisting in distinguishing monocytes/macrophages from other cells.
• Ly6G: Used to identify neutrophils and to separate these from Ly6C^+ monocytes.
• Gr-1 (RB6-8C5): Sometimes used, but recognizes both Ly6C and Ly6G; thus, using Ly6G and Ly6C separately provides more precise discrimination.

Typical applications are flow cytometry panels where combinations such as Ly6C (HK1.4), CD11b, F4/80, and Ly6G are used to map myeloid cell populations in blood, spleen, or tissue.

These combinations allow researchers to:

• Separate classical, non-classical, and inflammatory monocytes.
• Distinguish monocytes from neutrophils and other granulocytes.
• Study cell differentiation, immune activation status, and responses to inflammation or infection.

In summary, HK1.4 is most often paired with CD11b, F4/80, Ly6G, CD45, CD3, CD8, and CD4 for comprehensive immune cell profiling in mice.

Clone HK1.4 is a widely used monoclonal antibody targeting mouse Ly-6C (Ly6C1), and its key findings in scientific literature center on its applications in immunology, particularly for identifying and characterizing immune cell subsets.

Primary Applications

Clone HK1.4 has been extensively utilized across multiple experimental techniques. The antibody is employed in flow cytometry, immunohistochemistry, and in vitro activation assays for studying immune cell populations. Its versatility extends to in vitro activation studies, where it serves as a valuable tool for investigating immune cell behavior and function.

Specificity and Compatibility

A notable finding regarding clone HK1.4 is its compatibility with other commonly used antibodies. Specifically, HK1.4 does not block the binding of clone RB6-8C5, another widely used antibody in the field. This non-interference property makes it particularly useful for multi-marker flow cytometry experiments where researchers need to simultaneously detect multiple cell surface markers. Unlike the 1A8 clone, which shows interference with RB6-8C5, HK1.4 maintains compatibility, allowing researchers to use both antibodies in combination studies.

Target Molecule

The antibody specifically recognizes Ly-6C (Ly6C1), an important cell surface marker used to distinguish different subsets of immune cells, particularly monocytes and lymphocytes in mice. This specificity has made clone HK1.4 an essential reagent for immunological research requiring precise characterization of immune cell populations.

Dosing regimens for clone HK1.4, an anti-mouse Ly-6C antibody, exhibit significant variability depending on the experimental context and mouse model being studied. While specific published dosing protocols for HK1.4 are limited in the available literature, the general principles governing in vivo antibody dosing in mouse models provide insight into how such regimens are determined and adjusted.

General Dosing Considerations

Clone HK1.4 is a rat monoclonal antibody that recognizes mouse Ly-6C, a surface marker expressed on various myeloid cell populations including monocytes, macrophages, and certain granulocyte subsets. The antibody is typically supplied at a concentration of 0.5 mg/ml and requires titration for optimal performance in each specific application.

Factors Influencing Dosing Variations

The variability in HK1.4 dosing regimens stems from several key factors that differ across mouse models. The specific experimental objective—whether for cell depletion, functional blockade, or phenotypic analysis—fundamentally determines the dosing approach. Different mouse strains may also require adjusted doses due to variations in body weight, immune cell populations, and antibody clearance rates.

The disease model context significantly impacts dosing decisions. For example, tumor models, infection studies, and autoimmune disease models each present unique challenges in terms of target cell accessibility, disease kinetics, and the desired duration of antibody effect. Similar antibodies used for myeloid cell depletion, such as RB6-8C5 (anti-Gr-1, which recognizes both Ly6G and Ly6C), typically employ doses of 200-250 μg per mouse administered intraperitoneally every 2-3 days for sustained depletion.

Comparative Dosing Across Applications

In broader immunotherapy contexts, antibody dosing in mice follows established patterns that likely inform HK1.4 usage. Immune cell-depleting antibodies generally require 100-250 μg per mouse administered 2-3 times per week via intraperitoneal injection. For instance, CD4 and CD8 T cell depletion studies use 100 μg per dose every 4 days, while other protocols employ 200-250 μg doses.

When considering myeloid-targeted therapies more broadly, higher doses may be employed. Studies using anti-CSF-1 antibodies for myeloid cell modulation have utilized initial doses of 1 mg per mouse, followed by maintenance doses of 0.5 mg, demonstrating that myeloid-targeted interventions sometimes require more substantial antibody quantities.

Route and Timing Adjustments

The route of administration remains relatively consistent across most in vivo antibody studies, with intraperitoneal injection serving as the standard approach due to its ease of administration and reliable systemic distribution. However, the timing between doses varies considerably based on antibody clearance kinetics and the regeneration rate of target cell populations.

For sustained effects, dosing intervals typically range from every 2-3 days to weekly administrations, with more frequent dosing required when complete and continuous target cell depletion is necessary. The specific model's disease progression timeline also dictates whether pretreatment doses are needed or whether therapy begins concurrent with disease induction.

Model-Specific Optimization

Ultimately, each research application requires empirical optimization of HK1.4 dosing. Researchers must consider the baseline expression levels of Ly-6C in their specific mouse strain, the kinetics of the biological process under investigation, and whether the goal is transient modulation or sustained depletion of target populations. Pilot studies with dose-response curves and time-course analyses are recommended to establish the optimal regimen for each unique experimental context.