Anti-Ly49C (Clone 4LO3311) – Purified in vivo GOLD™ Functional Grade

The clone 4LO3311 is commonly used for in vivo applications in mice, particularly targeting the Ly49C receptor, which is expressed on NK cells, uterine NK cells, NKT cells, and CD8+ T cells. The antibody is typically used in functional assays, flow cytometry, immunoprecipitation, and inhibition assays. Here are some specific applications:

1. Inhibition of NK Cell Activity: The antibody can inhibit NK cell activity, which is useful for studying immune responses and cancer immunotherapy in mice models.

2. Immunotherapy and Oncology: It can be used to deplete or modulate specific immune cells, such as NK cells, which are involved in tumor surveillance and immune responses against cancer cells in mouse models.

3. Functional Studies: Since it binds to Ly49C, a receptor involved in immune regulation, the antibody helps in understanding the role of these receptors in immune responses in vivo.

4. Flow Cytometry: For identifying and quantifying cells expressing Ly49C, this antibody is used in flow cytometry to analyze immune cell populations in mice.

5. In Vivo Research: Generally, for any in vivo research that involves studying the function or depletion of Ly49C-expressing cells in mice.

The antibody 4LO3311 is a monoclonal antibody specific for murine Ly49C, a receptor found on subsets of mouse natural killer (NK) cells. In the literature, experiments often combine 4LO3311 with other antibodies or proteins to phenotype NK cells or study NK cell receptor expression.

Commonly used antibodies and proteins alongside 4LO3311 include:

• Other Ly49 family antibodies: To differentiate subsets of NK cells, studies frequently use panels including antibodies against Ly49A, Ly49D, Ly49G2, and Ly49I in addition to 4LO3311 (Ly49C), as the Ly49 receptor family members have overlapping but distinct expression patterns on NK cells.
• CD3 and NK1.1: These are standard markers to define NK cells (CD3− NK1.1+ cells), often used together with Ly49 antibodies. CD3 is a T cell marker, while NK1.1 is a pan-NK cell marker in certain mouse strains.
• CD49b (DX5): Used as another pan-NK cell marker, particularly in strains where NK1.1 is not expressed.
• CD226/DNAM-1: Sometimes used for functional or activation studies involving NK cells.
• Secondary antibodies: Depending on the detection system (e.g., flow cytometry or immunohistochemistry), secondary antibodies with fluorochrome or enzyme conjugates (e.g., FITC, PE, HRP) are used to visualize binding.

These combinations allow researchers to:

• Distinguish NK cell subsets by their Ly49 receptor profiles.
• Study functional differences or activation status using additional markers such as CD107a (degranulation), IFN-γ (cytokine production), or activation/inhibition markers.

In summary, other Ly49 antibodies, core NK cell markers (CD3, NK1.1, CD49b), and activation/functional antibodies are most often used alongside 4LO3311 in the literature on NK cell immunophenotyping and functional studies.

Key Scientific Findings Related to Anti-Ly49C Clone 4LO3311

Epitope Specificity and Staining Challenges

• 4LO3311 is a monoclonal antibody specifically directed against Ly49C (Killer cell lectin-like receptor 3, KLRA3; NK2.1) and binds an epitope located within a 32-amino acid region of the receptor.
• Staining of Ly49C using 4LO3311 is impacted by in vivo interactions with MHC class I molecules: In B6 mice, where Ly49C engages self-MHC class I molecules (H-2Kb), 4LO3311 fails to stain NK cells, likely due to epitope masking by cis-interactions between Ly49C and MHC class I on the same cell.
• Acid treatment restores binding: This masking effect can be reversed by acid treatment, which disrupts the interaction between Ly49C and MHC class I, allowing 4LO3311 to detect the receptor.
• Differential staining across mouse strains: While 4LO3311 does not stain B6 NK cells under normal conditions, it weakly stains NK cells from Cmv1r mice (which have a different MHC background) and strongly stains NK cells from B6 β2m knockout mice, which lack MHC class I expression.

Structural Insights and Functional Implications

• 4LO3311 and viral ligand m157 G1F bind the same stalk region of Ly49C: Both reagents require the receptor to adopt an extended configuration for binding, but structural constraints imposed by cis-binding to MHC class I prevent simultaneous binding of both the antibody and the viral ligand.
• Competitive binding dynamics: When Ly49C is engaged by MHC class I in cis, neither 4LO3311 nor m157 G1F can bind, which has implications for the detection and functional analysis of Ly49C+ NK cells in different experimental contexts.
• Implications for NK cell education and licensing: While the literature does not directly report on 4LO3311’s use in NK cell education studies, the antibody’s specificity and the challenges in staining underscore the complexity of studying Ly49C’s role in NK cell self-tolerance and licensing, as epitope accessibility is modulated by MHC class I interactions.

Technical and Availability Issues

• 4LO3311 is noted as non-commercially available and difficult to obtain: Researchers have reported unsuccessful attempts to acquire aliquots from multiple groups, highlighting limited access to this reagent for the scientific community.
• Alternative antibodies exist: Due to these challenges, other antibodies (e.g., 5E6, which recognizes both Ly49C and Ly49I) are often used, but these lack the strict specificity of 4LO3311, complicating the discrimination of Ly49C+ from Ly49I+ NK cells.

Summary Table: Key Features of 4LO3311 Citation Findings

Conclusion

Clone 4LO3311 is a highly specific anti-Ly49C antibody whose utility is limited by epitope masking when Ly49C engages MHC class I in cis, a challenge that can be overcome with acid treatment. Its restricted availability and the complexity of Ly49C-MHC interactions complicate its use in NK cell research, but it remains a valuable tool for dissecting Ly49C-specific functions when accessible. These findings highlight the importance of considering receptor configuration and ligand engagement when interpreting antibody-based assays in immunology.

The search results do not provide specific information on the dosing regimen of clone 4LO3311 across different mouse models. Therefore, I can only offer general guidance on how dosing regimens might vary depending on the context and application of antibodies in mouse models.

General Considerations for Antibody Dosing in Mouse Models:

1. Target and Species: The target antigen (e.g., PD-1, PD-L1, CTLA-4) and the species (mouse) are crucial for determining the dose. Different antibodies have different optimal dosing ranges based on their targets. For example, anti-PD-1 antibodies like RMP1-14 are typically used at 200-500 μg per mouse, while anti-PD-L1 antibodies like 10F.9G2 use 100-250 μg per mouse.

2. Route of Administration: The most common route for antibody administration in mouse models is intraperitoneal (i.p.) injection, as it is effective and easy to perform.

3. Dosing Schedule: The frequency of dosing can vary. For instance, anti-PD-1 antibodies might be administered every 3-4 days, while anti-PD-L1 antibodies might be given 2-3 times per week.

4. Applications: The specific application (e.g., cancer immunotherapy, infection models) can influence the dosing regimen. For example, combinations of anti-PD-1 and anti-CTLA-4 antibodies are common in cancer immunotherapy studies.

5. Experimental Design: The dosing regimen can be adjusted based on experimental goals, such as inducing desired immune responses or managing side effects.

Without specific data on clone 4LO3311, it's essential to consult the relevant scientific literature or the manufacturer's guidelines for precise dosing recommendations.