Anti-Mouse CD24 (Clone M1/69) – Purified in vivo PLATINUM^TM Functional Grade

Clone M1/69 is a rat monoclonal antibody commonly used in vivo in mice to detect and manipulate cells expressing CD24 (Heat Stable Antigen, HSA), with applications most notably in immunology and hematopoietic cell research.

Key in vivo applications include:

• Flow Cytometry and Cell Sorting: M1/69 is widely used to identify, quantify, and isolate CD24+ cell populations in mouse tissues, including B cells, dendritic cells, granulocytes, erythrocytes, and various progenitors, particularly in the bone marrow and spleen.
• Immunophenotyping: The antibody enables delineation of immune cell subsets and the mapping of immune cell development, notably stages of B lymphopoiesis and thymocyte maturation, by resolving the differential expression of CD24 during hematopoietic differentiation.
• In Vivo Cell Depletion or Blocking Studies: Functional-grade M1/69 (low-endotoxin, azide-free) can be injected into mice to block or deplete CD24+ cells, making it valuable for probing the role of CD24 in immune responses, development, autoimmunity, and in models of malignancy.
• Disease Modeling: Because CD24 is overexpressed in some mouse and human tumors, in vivo M1/69 can be used to study cancer cell biology, metastasis, and immune regulation in the tumor microenvironment.
• Adhesion and Signaling Studies: M1/69 can be used to explore the involvement of CD24 in cell adhesion (e.g., via interactions with P-selectin) and intracellular signaling during physiological and pathological processes.

Additional context and caveats:

• Use of M1/69 is essential for accurate immunophenotyping, as its binding can be affected by cell-type-specific glycosylation; consistency of clone usage is necessary for reproducible results.
• Different antibody formats (purified, functional grade, or conjugated with fluorochromes) are available for in vivo use, including variants specifically tested for low endotoxin content to minimize immune artifacts in animal experiments.
• In vivo applications are complemented by in vitro uses (e.g., immunohistochemistry, Western blot), but for direct animal studies, functional, clinical-grade antibody is recommended.

In summary, clone M1/69 is a standard tool for in vivo characterization and manipulation of mouse CD24+ cells, central to studies of immune cell differentiation, cancer biology, and immune regulation in murine models.

The M1/69 antibody, which targets murine CD24 (Heat Stable Antigen, HSA), is most commonly used in immunophenotyping panels to delineate murine hematopoietic cell populations. In the literature, M1/69 is frequently used in combination with a core set of other antibodies or markers, depending on the cell type and system under study.

Commonly used antibodies/proteins with M1/69 include:

• CD3: For identifying T cells.
• CD4 and CD8: To distinguish T helper and cytotoxic T cell subsets.
• CD19 or B220 (CD45R): For B cell identification.
• CD11b and Gr-1: For myeloid lineage cells, especially in bone marrow or splenic analyses.
• CD11c: To mark dendritic cells.
• CD44 and CD62L: For analyzing T cell activation and differentiation status.
• CD25 and CD69: As activation markers on T cells.
• Ter119: For erythroid lineage cells.
• Thy1 (CD90): For T cell and some stem/progenitor cell populations.
• CD45: A pan-leukocyte marker, often used to distinguish hematopoietic cells from others.

Further context and representative rationale:

• When M1/69 (anti-CD24) is used in flow cytometry to study B cell development, it is often combined with CD19/B220 and IgM/IgD to distinguish pro-B, pre-B, immature, and mature B cell stages.
• In myeloid cell or dendritic cell profiling, M1/69 is commonly partnered with CD11b, Gr-1, or CD11c to help distinguish subpopulations.
• In thymocyte subset analysis, M1/69 is used with CD4 and CD8 antibodies to resolve double negative, double positive, and single positive thymocyte populations.
• For hematopoietic stem and progenitor cell studies, a cocktail including c-Kit (CD117), Sca-1, CD34, and lineage markers such as CD3, B220, Gr-1, and Ter119 is commonplace, with M1/69 sometimes included to further refine lineage exclusion or inclusion.

Alternative clones sometimes used for CD24 staining in mice (instead of or alongside M1/69) include J11d and 30-F1, which may give subtly different staining results on certain lymphocyte subpopulations and thus are sometimes used for comparative purposes.

Summary Table of Common Partners:

M1/69 is a central marker in murine immunology, and is rarely used alone—its primary value is as part of multi-color flow cytometry panels where it helps delineate complex hematopoietic subsets in conjunction with the markers listed above.

Key Findings from Clone M1/69 in Scientific Literature

Recognition and Specificity

• Clone M1/69 is a rat monoclonal antibody widely used to detect mouse CD24, also known as the heat-stable antigen (HSA). It is primarily employed for immunophenotyping murine hematopoietic cells via flow cytometry, immunohistochemistry, and immunofluorescence.
• CD24/HSA is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, expressed on various cell types including erythrocytes, thymocytes, peripheral lymphocytes, and myeloid lineage cells.
• The antigen recognized by M1/69 is encoded by a surprisingly small gene—after signal peptide cleavage, the mature protein is only about 30 amino acids long. However, its molecular mass (35–50 kDa) is largely due to extensive N- and O-linked glycosylation, which varies by cell type and is crucial for its functional specificity.

Functional and Biological Insights

Cell Type Profiling and Isolation

• M1/69 is a central tool for profiling and isolating murine immune cell populations, including dendritic cells, myeloid cells, and thymocytes, due to its specificity for CD24/HSA.
• Expression dynamics: CD24 expression is high during early B-cell development (from pro-B cells to mature B cells), but declines or is absent on plasma cells. It is also present on many B-cell malignancies, making it a valuable marker for studying B-cell development, leukemia, and lymphoma.
• Functional roles: CD24 is implicated in regulating B-cell proliferation, maturation, and autoimmunity. It also interacts with P-selectin (CD62P), suggesting a role in cell adhesion and signaling.

Glycosylation and Molecular Heterogeneity

• The extensive and variable glycosylation of CD24 results in molecular heterogeneity, which can influence antibody staining patterns across different cell lineages.
• Differential glycosylation is thought to confer cell-type-specific functions, as the carbohydrate moieties rather than the peptide backbone are primarily responsible for the antigen’s biological activity.

Technical and Comparative Considerations

• Subtle staining differences may be observed between anti-CD24 clones (e.g., M1/69, J11d, 30-F1), highlighting the importance of consistent antibody usage in research.
• Applications: M1/69 is validated for flow cytometry, Western blotting, immunohistochemistry, and functional assays in both in vitro and in vivo settings.
• Recommendations: Careful titration is advised for optimal performance, especially in flow cytometry.

Summary Table: Key Attributes of Clone M1/69

Conclusion

Clone M1/69 is a foundational reagent in immunology and cell biology for the study of mouse CD24/HSA. Its utility lies in its specificity for a variably glycosylated, developmentally regulated cell surface marker that is critical for immune cell identification, isolation, and functional analysis. The literature emphasizes the importance of glycosylation in conferring functional diversity to CD24 and the need for careful methodological consistency when using this clone in research.

Dosing Regimens of Clone M1/69 (Anti-Mouse CD24) in Mouse Models

Variability in Dosing Protocols

There is no standardized dosing regimen for clone M1/69 (anti-mouse CD24 antibody) across mouse models; both the dose and frequency are typically tailored to the specific experimental goals, the sensitivity of the mouse strain, and the outcomes being assessed. Published protocols and vendor details do not provide a universal recommendation, reflecting the heterogeneity in experimental designs and biological endpoints in immunology research.

Factors Influencing Dosing

• Experimental Objectives: The amount and frequency of M1/69 administration depend on whether the goal is to deplete CD24+ cells, block CD24 function, or use the antibody as a marker for cell identification and sorting.
• Mouse Strain Sensitivities: Different strains may exhibit varying responses to antibody treatments due to genetic background, immune status, or baseline CD24 expression levels.
• Route of Administration: Intraperitoneal (IP), intravenous (IV), or other routes may be used, each influencing pharmacokinetics and thus effective dose.
• Duration of Study: Acute versus chronic dosing schedules are designed based on whether short-term effects or long-term immunological changes are being studied.

Published Evidence and Vendor Guidance

Vendor product descriptions and available literature emphasize that dosing regimens "vary significantly across studies and mouse models," and researchers are advised to design their protocols based on pilot experiments and literature precedents relevant to their specific model and question. No single example or table of doses is provided by vendors or in summary publications, highlighting the need for empirical optimization in each experimental context.

Parallels with Monoclonal Antibody Pharmacokinetics

While M1/69 is not typically used as a therapeutic, monoclonal antibodies (mAbs) in mice generally exhibit linear pharmacokinetics, with parameters (clearance, volume of distribution) that are similar across different targets and within a species. This suggests that, while dosing may be tailored, the underlying pharmacokinetic principles are consistent—meaning that established mAb dosing strategies in mice could theoretically be adapted for M1/69, with adjustments for target expression and desired biological effect. However, published data specifically on M1/69 pharmacokinetics or head-to-head dosing comparisons across models are lacking.

Practical Recommendations

Given the lack of a universal protocol, researchers should:

• Consult prior literature using M1/69 in similar mouse models and for analogous experimental endpoints.
• Perform pilot dose-finding studies to establish efficacy and tolerability in their specific system.
• Monitor outcomes (e.g., CD24+ cell depletion, functional assays, health monitoring) to iteratively refine the regimen.

Summary Table: Key Considerations

Conclusion

Dosing regimens for clone M1/69 vary widely across mouse models and studies, with no single protocol mandated by vendors or the literature. Effective use requires careful consideration of experimental context, strain differences, and empirical optimization, often informed by prior publications in similar systems. Systematic, model-specific pilot experiments remain the gold standard for establishing appropriate dosing.