Anti-Mouse/Human CD45R (B220) [Clone RA3-6B2] — Purified in vivo GOLD™ Functional Grade

Common In Vivo Applications of Clone RA3-6B2 in Mice

Clone RA3-6B2 is widely used in immunology research for its ability to target the CD45R/B220 isoform, which is predominantly expressed on mouse B cells. Here are some common in vivo applications of this clone:

1. B Cell Depletion and Immunomodulation: The RA3-6B2 antibody is commonly used for in vivo B cell depletion and to modulate B cell responses. This is particularly useful in studies aimed at understanding B cell functions and immune regulation.

2. Immunological Research Models: RA3-6B2 is applied in various mouse models to study autoimmune diseases, infection responses, and cancer by manipulating B cell populations and functions.

3. Flow Cytometric Analysis: Although often used ex vivo, RA3-6B2 can be applied in conjunction with flow cytometry to analyze B cell populations in tissues obtained from mice following in vivo treatments.

4. In Vivo Modulation of Disease Models: This includes models for autoimmune diseases and infections, where altering B cell responses can provide insights into disease mechanisms and potential therapeutic interventions.

5. Immunotherapy and Immune Tolerance Studies: RA3-6B2 can be used to study immune tolerance and the impact of B cell depletion on immune responses in mice, facilitating research on immunotherapeutic strategies.

Overall, clone RA3-6B2 is a versatile tool in mouse models for studying B cell biology and immune regulation in vivo.

To profile immune cell populations in the literature, especially in mouse immunology, the RA3-6B2 antibody (specific for CD45R/B220, a pan–B cell marker) is frequently paired with antibodies targeting other key cell surface proteins to enable comprehensive identification and characterization of immune subsets.

Commonly Paired Antibodies and Proteins

Additional Context

• B220 (RA3-6B2) is a pan–B cell marker but is also expressed on some non-B cells (e.g., activated T cells, NK progenitors, and LAK cells), so co-staining with CD19 increases specificity for bona fide B cells.
• CD4 and CD8 are used to identify T cell subsets and to distinguish them from B cells in mixed lymphocyte populations.
• NK1.1 is used to exclude NK cells, and CD11b for excluding myeloid cells, both of which might express B220 in certain contexts.
• IgM and IgD are classic markers for mature B cells, while CD21 and CD23 help distinguish follicular, marginal zone, and other B cell subsets.

Typical Multicolor Flow Cytometry Panels

A typical mouse lymphocyte panel might include:

• RA3-6B2 (B220) and CD19 for B cells
• CD3, CD4, CD8, and NK1.1 for T and NK cells
• CD11b for myeloid exclusion
• IgM, IgD, CD21, CD23 for B cell subsetting

These combinations allow researchers to accurately identify, subset, and analyze B cell populations and to distinguish them from other immune lineages.

Summary

RA3-6B2 is most commonly used in combination with CD19, CD3, CD4, CD8, NK1.1, CD11b, IgM, IgD, CD21, and CD23 in the literature to thoroughly characterize immune cell populations, especially in mouse models. These combinations are standard in flow cytometry and immunohistochemistry panels for comprehensive immune phenotyping.

Clone RA3-6B2 is one of the most widely used monoclonal antibodies targeting the CD45R/B220 isoform and is heavily cited in scientific literature for identifying and characterizing mouse B cells, as well as certain T cell and NK cell populations.

Key findings and applications from RA3-6B2 citations include:

• B Cell Marker: RA3-6B2 is the standard marker for murine B lymphocytes at all developmental stages from pro-B to mature and activated B cells, though its expression is decreased on plasma cells and certain memory B cell subsets. RA3-6B2 binding is developmentally regulated during B cell maturation.

• Functional Studies and Immune Modulation:

  • RA3-6B2 is used to track B cell development, activation, and differentiation in studies of immune responses, autoimmune models, and hematopoietic lineage tracing.
  • It has functional effects: the antibody has been reported to enhance isotype switching in vitro and inhibit in vivo B cell responses, making it a tool for immune modulation experiments.

• Breadth of Reactivity:

  • While predominantly identifying B cells, RA3-6B2 also reacts with some T cell subsets (including activated T cells), NK cell subsets, and lymphocyte precursors in bone marrow and fetal liver.
  • Notably, cross-reaction has been observed with some activated human T lymphocytes.

• Epitope Specificity:

  • RA3-6B2 binds to an epitope on the extracellular domain of CD45R (dependent on exon A expression and certain carbohydrate modifications). CD45 is a protein tyrosine phosphatase important in immune receptor signal transduction.

• Research Citations:

  • RA3-6B2 is cited in research investigating immune cell composition in tumors and the effects of therapies (e.g., checkpoint inhibition and immune responses in mouse models). It has been pivotal in studies of immune clonality, B and T cell subsets, and functional lymphoid cell diversity.
  • Used as a quality control or reference for flow cytometry, immunohistochemistry, and related techniques due to its specificity and broad adoption.

• Limitations:

  • CD45R/B220 is not perfectly B cell-specific (unlike CD19), as it can be expressed by certain NK, T, and progenitor cells, so careful gating and controls are necessary in immunophenotyping.

Overall, the RA3-6B2 clone is heavily cited for its critical role as a pan-B-cell marker in murine models, its utility in immune modulation, and its centrality in studies of hematopoietic and immune system biology.

Dosing regimens for clone RA3-6B2, a monoclonal antibody targeting mouse CD45R/B220, vary significantly depending on the experimental purpose, mouse strain, and application, but for flow cytometry, the recommended dose is typically ≤0.5 µg per test, while for in vivo modulation, the dose and frequency must be empirically determined for each model.

• Flow Cytometry and Ex Vivo Applications:

  • The most common regimen for flow cytometry is up to 0.5 µg antibody per test, which usually refers to staining a sample with 10^5 to 10^8 cells in 100 µL volume. Users are advised to titrate the antibody for optimal staining in their specific assay and mouse model.
  • This dosage typically applies across different strains including C57BL/6, BALB/c, and others unless there is a known alteration in CD45R expression or epitope masking within the model.

• In Vivo Modulation or Depletion Studies:

  • RA3-6B2 has been reported to modulate B cell responses and affect both T and B cell differentiation when administered in vivo, but optimal dosing regimens are not standardized and must be empirically determined for the particular mouse model and experimental aim (e.g., B cell depletion, functional modulation).
  • Published protocols sometimes use regimens similar to other depletion antibodies (e.g. 200–250 µg/mouse via intraperitoneal injection every 2–3 days for anti-CD8 or anti-Gr-1 antibodies), but there is no universal dose-for-effect for RA3-6B2 itself, and companies supplying the antibody recommend laboratory-specific optimization.

• Factors Influencing Dose Selection:

  • Mouse Model: Strains with altered immune compartments (e.g., lpr mice, myeloproliferative models) may require modified dosing due to differences in CD45R expression or immune system activity.
  • Application: For immunohistochemistry and tissue staining, dilution factors and concentrations differ depending on tissue fixation methods (acetone-fixed, paraffin-embedded, etc.).
  • Epitope Accessibility: The antigen's expression level and masking by other molecules (such as in activated T cells or NK cell progenitors) may necessitate dose adjustments.

• Manufacturer and Literature Guidance:

  • All major commercial suppliers emphasize empirical titration of RA3-6B2 for each mouse model and experimental setting, due to possible variability in antigen density and tissue type.
  • Literature describing in vivo effects—such as B cell depletion or immune modulation—rarely provides a universal protocol, instead noting results at specific doses and inviting further optimization.

In summary, clone RA3-6B2 is most commonly used at ≤0.5 µg/test for flow cytometry, but in vivo regimens require empirical optimization depending on mouse strain and experimental goals. No standardized dosing exists for in vivo depletion or modulation: researchers must titrate for their specific mouse model and desired outcome.