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

Clone RA3-6B2 is a monoclonal antibody targeting mouse CD45R (also known as B220), and in in vivo mouse studies, it is used primarily to deplete or modulate B cells and influence immune cell differentiation and function.

Key uses and details in in vivo studies:

• B cell depletion or modulation: RA3-6B2 administration in mice can significantly reduce the number of B cells, as well as affect T cell populations.
• Functional modulation: It is reported to alter B cell and T cell development, differentiation, and immune responses (such as antibody levels, including pathogenic anti-DNA antibodies in autoimmune disease models).
• Immunological disease models: RA3-6B2 has been used in autoimmune-prone strains (like MRL/lpr mice) to reduce autoantibody production and lymphadenopathy.
• Marker in in vivo tracking and depletion studies: Besides depletion, RA3-6B2 can also help temporarily label and track B220+ cells for cell fate and migration studies.

Mechanism: The antibody binds to an extracellular epitope of the CD45R molecule (predominantly B cells, but also certain T and NK cell subsets), leading to cell depletion via Fc-dependent mechanisms or modulating their response and function in vivo.

Experimental applications:

• Induction or suppression of immune responses
• B cell ablation in mechanistic studies of immune function
• Therapeutic modulation in autoimmune disease models
• In vivo cell tracking and immune subset characterization via flow cytometry or imaging following antibody tagging

References and usage precautions:

• RA3-6B2’s effects can vary by dose, administration route, and model; published studies cited by antibody suppliers provide protocols and references for specific applications.
• As it can also label subsets of T and NK cells, experimental design should consider possible off-target effects.

In summary, clone RA3-6B2 is widely used for in vivo B cell depletion and immunomodulation in mouse models, and plays a critical role in elucidating B cell functions and immune regulation.

RA3-6B2 is a monoclonal antibody widely used for detecting the CD45R/B220 isoform, primarily as a murine B cell marker. In the literature, RA3-6B2 is frequently used in combination with the following antibodies or proteins, depending on the experimental context:

• CD19: Another pan-B cell marker, often co-stained with RA3-6B2 to help distinguish between different B cell subsets.
• CD3: A T cell marker, frequently used alongside RA3-6B2 to differentiate T cells from B cells in mixed lymphocyte populations.
• CD4 and CD8: Used to further delineate T cell subsets in conjunction with B cell gating.
• CD11b or Mac-1/Gr-1: Myeloid lineage markers to exclude or identify myeloid cells when characterizing splenocyte or bone marrow populations.
• IgM and IgD: Immunoglobulin isotypes to identify B cell maturation stages when combined with B220 staining.
• NK1.1 (CD161) or DX5: Natural killer (NK) cell markers, as B220 is also expressed on certain subsets of NK and T cells.
• CD21 and CD23: To distinguish follicular versus marginal zone B cells.

These combinations are common in multicolor flow cytometry panels, immunohistochemistry, and immunofluorescence assays to characterize lymphocyte subsets in mouse models. Specific references and applications, such as spatial biology and tissue staining, are also documented.

In summary, antibodies against CD19, CD3, CD4, CD8, NK1.1, CD11b, IgM, IgD, CD21, and CD23 are among the most frequently paired with RA3-6B2 in the literature to thoroughly profile immune cell populations.

Clone RA3-6B2 is principally recognized in scientific literature as a monoclonal antibody specific for the CD45R/B220 isoform of CD45, and it is extensively used as a marker for B cells, certain T cell subsets, and NK cell subsets in both mouse and, less commonly, human research. Key findings from its citations highlight the following:

• Molecular and Cellular Target:

  • RA3-6B2 targets the extracellular domain of CD45R/B220, with binding that depends on the expression of exon A and specific glycosylation patterns.
  • CD45R is a 220 kDa glycoprotein involved in the regulation of immune cell activation, differentiation, and signaling by modulating protein tyrosine phosphatase activity.

• Application as a Marker:

  • It is widely used to identify B cell lineage (pro-B to mature B cells) and also marks lytically active NK cell subsets and non-MHC-restricted cytotoxic T lymphocytes.
  • The antibody is used in various flow cytometry and immunohistochemistry protocols, including on frozen and paraffin-embedded tissue sections.
  • Due to its ability to distinguish B lineage cells from other hematopoietic populations, RA3-6B2 is fundamental in studies requiring B cell isolation, tracking, or depletion.

• Functional Studies:

  • Studies using RA3-6B2 have enabled modulation of B cell responses in vitro and in vivo, and it has served in immunoprecipitation assays to study CD45 isoforms and glycosylation-mediated regulation.

• Biological Insights:

  • Research with RA3-6B2 has contributed to understanding the roles of CD45 isoform diversity in cell signaling and immune regulation, specifically in B and T cell antigen receptor signaling and cytokine receptor signaling via JAK kinase pathways.
  • The heavy glycosylation of CD45R gives rise to its higher molecular weight in B cells (hence the designation B220), further underscoring its value in distinguishing B cell populations.
  • RA3-6B2’s recognition epitope and carbohydrate dependency have been important in dissecting the structure–function relationships of CD45R isoforms in immune cell biology.

• Technical and Cross-reactivity Notes:

  • RA3-6B2 has demonstrated variable reactivity across species, predominantly used in mice, but with limited application in human systems due to differences in CD45 isoform expression.
  • Conjugated forms (e.g., FITC, APC, DyLight 488) facilitate multiplexed immunophenotyping in advanced cytometry and single-cell biology platforms.

In summary, RA3-6B2’s citations support its central role as a B cell marker and tool in immunological research, contributing to the understanding of immune cell identity, differentiation, and signaling pathways critical for adaptive immunity.

Dosing regimens of clone RA3-6B2 (anti-mouse CD45R/B220) vary primarily by application, tissue processing, and, to a lesser extent, mouse strain or model. The most common variable is dose per test in assays such as flow cytometry, with adjustments based on total cell number, tissue type, and experimental sensitivity requirements.

Key points on dosing regimens:

• Flow cytometry: The recommended dose for flow cytometric analysis using clone RA3-6B2 is typically ?0.5 µg per test, where a "test" is defined as the amount needed to stain the desired number of cells (commonly in a 100 µL volume, with 10? to 10? cells). Precise dosing may require titration for optimal results with specific mouse tissues or cell preparations.

• Immunohistochemistry (paraffin-embedded tissues): RA3-6B2 has been used for immunostaining of fixed, paraffin-embedded murine tissues. Protocols recommend initial titration to determine the appropriate dilution, as fixation and embedding can alter antigen exposure, impacting required dose.

• Model-Specific Factors: The literature and datasheets caution that antibody reactivity and working conditions may vary between different mouse strains or models, thus experimental optimization is advised for each context. Some mutant mouse lines (such as lpr/lpr), or cells at particular activation stages, may express CD45R/B220 differently, hence potentially affecting dosing needs.

Additional considerations:

• Cross-reactivity to other antigens (e.g., in knockout or transgenic lines) is rare but may require further assay titration.
• Fluorophore-conjugated forms (FITC, APC, etc.) may have slightly different optimal doses due to signal-to-noise considerations but are generally comparable to unconjugated forms in recommended initial titrations.
• Titration and Controls: For any new mouse model or tissue, careful antibody titration and use of positive/negative controls are essential to determine the optimal dosing regimen.

In summary:
Dosing of clone RA3-6B2 is generally standardized by assay type (e.g., ?0.5 µg/test for flow cytometry) rather than by mouse model, but empirical titration is necessary for each new tissue or model due to potential differences in antigen expression and tissue processing sensitivity.

No search results provided examples of dramatic regimen changes specific to particular disease models or mouse strains, so most variation centers on general optimization rather than fundamental dosage shifts.