Anti-Mouse/Human CD45R (Clone RA3-6B2) – Purified in vivo PLATINUM™ Functional Grade

Applications of Clone RA3-6B2 in In Vivo Mouse Studies

Clone RA3-6B2 is a widely used monoclonal antibody targeting CD45R (also known as B220), a key B cell surface marker in mice. Its applications in vivo are primarily focused on immunology research, particularly for manipulating B cell populations and functions.

Functional Uses

• B Cell Modulation: RA3-6B2 is directly administered to mice to modulate B cell responses. In vivo treatment with this antibody has been shown to reduce the number of both T and B cells and to modify their functional activity. This makes it useful for temporarily depleting or altering B cell populations in experimental models.
• Depletion and Lineage Analysis: It is sometimes used in combination with other lineage-specific antibodies to deplete specific hematopoietic populations in vivo, allowing researchers to study the roles of B cells and related cell types in immune responses, development, or disease models.
• Functional Studies: RA3-6B2 can be used to investigate the consequences of B cell modulation on immune responses, including antibody production, immune regulation, and pathogenesis in autoimmune or infectious disease models.

Mechanism of Action

Clone RA3-6B2 recognizes an epitope on the extracellular domain of the transmembrane CD45 glycoprotein, which is dependent on the expression of exon A and specific carbohydrate residues. Binding of this antibody can trigger signaling that leads to B cell depletion, altered activation, or functional modulation in vivo.

Practical Considerations

• Preparation: RA3-6B2 is available in various formats, including low-endotoxin versions for in vivo use, ensuring minimal off-target immune activation.
• Dosing: Optimal doses and treatment regimens should be determined empirically for each experimental protocol, as the effects can vary depending on the research context.
• Controls: Appropriate isotype controls are recommended to ensure that observed effects are due to specific targeting of CD45R rather than nonspecific antibody effects.

Summary Table

Conclusion

Clone RA3-6B2 is a versatile tool in mouse immunology, primarily used in vivo to deplete or functionally modulate B cells, thereby enabling researchers to dissect the roles of B cells in health and disease. Its specificity, availability in research-grade formats, and well-characterized effects make it a staple in preclinical immunological studies.

RA3-6B2 is commonly used as a B cell marker (CD45R/B220) in mice, and in the literature, it is often used in combination with antibodies or proteins that identify other immune cell lineages or distinguish B cell subsets. Some of the most frequently co-utilized markers include:

• CD3: A T cell marker, frequently used to exclude or gate out T cells in B cell identification or depletion studies.
• CD19: Another B cell–specific marker, often paired with B220 to more precisely define B cell populations.
• CD11b: A myeloid/monocyte/macrophage lineage marker that helps exclude or analyze myeloid cells in multi-color flow cytometry panels.
• Gr-1: Identifies granulocytes (e.g., neutrophils) and is often used for lineage exclusion.
• Ter-119: Labels erythroid cells, especially excluded in hematopoietic lineage depletion experiments.
• NK1.1: A marker for natural killer (NK) cells, used in multi-marker panels.

Combinations of these markers are used, for example, in lineage cocktails for "lineage-negative" gating strategies (Lin-) in the study of stem and progenitor cells, or for more refined analyses of B cell maturation and subsets.

Additionally, isotype controls (e.g., rat IgG2a) are used alongside RA3-6B2 for proper gating and background determination in flow cytometry and functional in vivo experiments.

In review:

• CD3, CD19, CD11b, Gr-1, Ter-119, NK1.1, and isotype controls are among the common antibodies/proteins used with RA3-6B2 in immunology research, especially in mouse hematopoietic and immune system analyses.

These combinations enable researchers to precisely identify, characterize, or deplete specific immune cell populations in diverse experimental settings.

Clone RA3-6B2 is a monoclonal antibody widely used in scientific research to detect and isolate the CD45R/B220 isoform of CD45, primarily on mouse B cells, certain T cell subsets, and NK cell subsets. The key findings from citations in scientific literature include:

• Specificity and Target: RA3-6B2 binds to a unique epitope on the extracellular domain of mouse CD45R/B220, which depends on the presence of exon A and specific carbohydrate residues on the glycoprotein. This makes it an essential tool for the identification and study of B lineage cells.

• Applications:

  • Flow cytometry: RA3-6B2 is one of the standard markers for murine B cells, widely used to sort or identify these cells in research.
  • Immunoprecipitation: Used to pull down the high molecular weight CD45 (220 kDa) from cell extracts.
  • Immunohistochemistry: Applicable for both frozen and paraffin-embedded tissue sections to visualize B cells in tissue contexts.
  • Functional modulation: RA3-6B2 can alter B cell function in vitro and in vivo, including modulating immune responses and possibly inhibiting B cell activity under certain experimental conditions.
  • Single-cell proteomics and spatial biology: Conjugated versions (e.g., TotalSeq) enable multi-omic profiling and spatial mapping of B cells at the single-cell level using protocols compatible with platforms like CITE-seq.

• Research Impact and Recognition:

  • The antibody is cited in studies exploring B cell development and activation, immune modulation, and the functional diversity of lymphocyte subsets.
  • RA3-6B2 is recognized for its specificity, cross-reactivity, and reliability for murine applications, with limited cross-reactivity to other species such as humans or cats.

• Product Variants and Citations:

  • Multiple vendors supply RA3-6B2 conjugated to various fluorochromes (e.g., FITC, APC) for use in different experimental setups, with more than a dozen primary literature citations for the antibody clone's applications, especially in immunology and cell biology research.

In summary, clone RA3-6B2 is an authoritative and versatile tool for mouse B cell identification, functional analysis, and immunological research, with broad validation in the literature for both basic and advanced applications such as flow cytometry, histology, and single-cell studies.

Dosing regimens for clone RA3-6B2 (anti-mouse CD45R/B220 antibody) primarily vary according to the mouse model application and experimental purpose (e.g., flow cytometry vs. in vivo depletion). The most common use is in flow cytometric analysis, with recommended doses of ?0.5 µg per test for staining mouse splenocytes. For tissue staining, titration is suggested for optimal performance, as cell density and tissue type affect antibody requirements.

• Flow Cytometry/Cell Sorting:

  • Dose: ?0.5 µg per test (in 100 µL volume, cell numbers 10?–10? per test).
  • Recommendation: Carefully titrate for each mouse strain and tissue/cell source for optimal signal-to-noise ratio.
  • Species: Used in C57BL/6, BALB/c, and lpr/lpr mutant mice, among others.
  • Note: RA3-6B2 binds the exon A-restricted isoform of CD45R/B220, highly expressed on the B-cell lineage but also found on select activated T cells, NK cell progenitors, etc..

• Tissue Immunohistochemistry:

  • Suitability: Used for paraffin-embedded tissues; optimal concentration may vary depending on fixation and tissue processing.
  • Recommendation: As above, titration required. Typical concentrations are not standardized across models and require empirical optimization.

• In Vivo Depletion/Functional Studies:

  • No standardized dosing regimens for RA3-6B2 in live mouse models were found in the available results. Commonly, depletion antibodies like anti-CD4 (GK1.5), anti-CD8 (2.43), or anti-Gr-1 (RB6-8C5) are used at 200–250 µg/mouse by intraperitoneal injection every 2–3 days. However, this guide does not list RA3-6B2 dosing for in vivo administration.

Key Considerations:

• Dosing variability arises from differences in:
  • Mouse strain and immune phenotype
  • Application (flow cytometry vs. immunohistochemistry)
  • Experimental purpose (cell selection, antibody depletion, etc.)
• Empirical titration is essential, as protocols may need adjustment for optimal performance in specific models and sample types.

Summary Table: RA3-6B2 Dosing by Application

RA3-6B2 dosing requires individual optimization per experiment and model. No fixed in vivo regimen is broadly accepted for RA3-6B2, unlike other depletion antibodies. For accurate dosing in a specific mouse model or application, consult product datasheets and empirical pilot studies.