Anti-Mouse CD172a [P84] – Purified in vivo PLATINUM™ Functional Grade

Common In Vivo Applications of Clone P84 in Mice

Clone P84 is a widely used rat monoclonal antibody targeting mouse CD172a (SIRPα), a receptor expressed predominantly on myeloid cells including monocytes, macrophages, dendritic cells, and some neurons. Its in vivo utility is supported by several key applications:

• Blockade of SIRPα–CD47 Interaction: P84 is commonly used to block the interaction between SIRPα and its ligand CD47 in vivo. This blockade inhibits the “don’t eat me” signal delivered by cancer cells to macrophages, thereby promoting macrophage-mediated phagocytosis of tumor cells—a strategy under investigation for cancer immunotherapy. The antibody has been shown to have neutralizing activity in both in vitro and in vivo settings.
• Modulation of Immune Cell Function: By blocking SIRPα, P84 can alter the behavior of myeloid cells, particularly in the context of immune responses and tumor microenvironment. This includes potential effects on dendritic cell migration and activation, as well as modulation of macrophage phagocytic activity.
• Research Tool in Cancer Immunotherapy Models: P84 is employed in preclinical mouse models to study the role of the SIRPα–CD47 axis in tumor immune evasion and to evaluate novel immunotherapies that target this pathway.
• Investigation of Neurological and Stem Cell Biology: Although less common, P84 may also be used to study SIRPα’s role outside the immune system, given its expression in neuronal tissue and stem cells.

Technical Considerations

• Isotype and Endotoxin: P84 is typically a rat IgG1, κ antibody, and is available in low-endotoxin formulations suitable for in vivo use.
• Dosing and Purity: For optimal performance, the antibody should be carefully titrated in each experimental system, and high-purity, low-endotoxin lots are preferred for in vivo studies.
• Control Experiments: Appropriate isotype controls should be included in experimental designs to ensure specificity of observed effects.

Summary Table

Clone P84 is thus a versatile tool for in vivo studies focused on the SIRPα–CD47 axis, particularly in cancer immunotherapy, immune modulation, and basic biology research in mice.

Commonly used antibodies or proteins alongside P84 (often referring to the anti-mouse CD172a [P84] antibody or the nuclear matrix protein p84 antibody) include several secondary reagents, cellular markers, and multiplexed targets for broader biological context or multiplex analysis.

Key examples from the literature include:

• Secondary Antibodies

  • Goat anti-Rabbit IgG (for detecting primary rabbit antibodies like P84 if used as primary).
  • Goat anti-Mouse IgG (when P84 is from mouse origin).

• Counterstains and Visualization Tools

  • Phalloidin (binds F-actin): Used as a cytoskeleton marker; often combined with nuclear or other markers for subcellular localization contrast.
  • DAPI: Stains DNA, enabling nuclear visualization in multiplex IF/IHC experiments.

• Multiplexed with Cancer or Proliferation Biomarkers

  • Ki-67: Cell proliferation marker.
  • p53, ER (Estrogen Receptor), PR (Progesterone Receptor), HER2: Common in multiplex IHC/IF for cancer profiling.

• Immunoprecipitation and Western Blot Controls

  • EasyBlot anti-rabbit IgG (HRP): Secondary for immunoprecipitation experiments.
  • Isotype controls: Such as mouse IgG2b for specificity validation in flow cytometry and WB.

• Functional/Pathway-Related Proteins
  For studies on PI3Kγ regulatory proteins:

  • p101: An alternative regulatory partner to p84 in PI3Kγ complexes, often analyzed in parallel for mechanistic studies.

• Other Applications and Cell Surface Markers

  • Additional surface antigens and lineage markers may be included depending on the study, e.g., CD11c for dendritic cells or other immune cell markers when characterizing cell populations with CD172a (P84).

Overview Table:

The exact set of co-used antibodies or proteins will vary based on experiment type (IHC, IF, IP, flow cytometry) and biological system under study.

Clone P84 is a rat monoclonal antibody that specifically recognizes CD172a (SIRPα - Signal Regulatory Protein alpha) in mice, and research using this antibody has revealed several important findings about SIRPα function and its potential therapeutic applications.

Molecular Characterization of P84/SIRPα

The P84 molecule was identified as being identical to SHPS-1, a member of the immunoglobulin superfamily. The full-length cDNA encodes a 509-amino-acid peptide with a calculated molecular weight of 56 kDa, though it appears larger on SDS-PAGE due to extensive glycosylation through 17 potential N-glycosylation sites. When treated with N-glycosidase F, the molecular weight shifts from 86 kDa to 64 kDa and from 77 kDa to 42-55 kDa, confirming the significant contribution of carbohydrate modifications. The protein exists in two forms generated by alternative splicing, with the smaller form lacking a 218-amino-acid segment.

The molecule contains a transmembrane domain and a cytoplasmic segment with multiple potential tyrosine phosphorylation sites at positions 436, 460, 477, and 501, which are crucial for its signaling function. These phosphorylation sites enable the recruitment and activation of tyrosine phosphatases SHP-1 and SHP-2 through immunoreceptor tyrosine-based inhibitory motifs (ITIMs).

Cancer Immunotherapy Applications

Research using clone P84 has demonstrated significant implications for cancer therapy. Studies comparing P84 with another anti-SIRPα antibody (MY-1) revealed important functional differences in blocking the CD47-SIRPα interaction. While MY-1 blocks CD47 binding and showed marked attenuation of tumor formation, P84 has little effect on the CD47-SIRPα interaction but still demonstrated a smaller yet significant inhibitory effect on tumor growth in RENCA renal cell carcinoma models.

When mice were treated with P84 after tumor establishment, the antibody showed reduced efficacy compared to early treatment, with delayed treatment having no significant effect on either tumor growth or survival. In metastatic models using B16BL6 melanoma cells, P84 was less effective than MY-1 in reducing lung metastatic nodules. These findings suggest that antibodies blocking the CD47-SIRPα interaction are more effective than those that don't, though P84 retains some tumor-suppressive activity through alternative mechanisms.

Cellular Expression and Function

Clone P84 has been extensively used to characterize SIRPα expression patterns on myeloid cells, including granulocytes, dendritic cells, macrophages, mast cells, and hematopoietic stem cells. The antibody has proven particularly useful in flow cytometric analysis of mouse bone marrow cells, where it helps identify CD11b+ myeloid populations expressing CD172a.

Studies have shown that SIRPα plays regulatory roles in several physiological processes, including inhibition of host cell phagocytosis by macrophages and bi-directional activation of T cells and dendritic cells. The protein is involved in negative regulation of receptor tyrosine kinase-coupled signaling pathways and responds to various stimuli including serum, growth factors, insulin, and cell adhesion signals.

Technical Applications

Clone P84 has been reported to have neutralizing activity and is widely used in flow cytometry applications. The antibody can be used at concentrations of 0.5 µg or less per test, with optimal detection using blue, green, or yellow-green lasers with excitation at 488-561 nm. It demonstrates specificity for SIRPα without cross-reactivity to SIRPβ family members, unlike some other anti-SIRPα antibodies.

The antibody is available in multiple formats including purified, PE-conjugated, and Ultra-LEAF preparations for in vivo functional studies. Clone P84 has become a standard research tool for investigating SIRPα biology and evaluating therapeutic strategies targeting the CD47-SIRPα axis in cancer and other diseases.

Dosing Regimens of Clone P84 (Anti-Mouse CD172a/SIRPα) in Mouse Models

Clone P84 is a monoclonal antibody targeting mouse CD172a, also known as Signal Regulatory Protein Alpha (SIRPα), and is widely used for in vivo experiments, flow cytometry, and functional studies in mice. The dosing regimens in mouse models vary depending on the experimental design, target tissue, and therapeutic or mechanistic goals.

Key Examples of Dosing Regimens

The available literature provides several specific examples of how P84 is administered in different models, with dosing that is tailored to the experimental context:

Neuroblastoma Tumor Model (NXS2)

• Dose: 20 μg per mouse per injection.
• Route: Intraperitoneal (i.p.) injection.
• Schedule: Injections on days 3, 7, 10, 14, 17, and 21 post-tumor inoculation (6 doses total).
• Purpose: Tested in combination with other therapies to assess anti-tumor effects.

Staining for Flow Cytometry

• Dose: ≤1.0 μg per 10⁶ cells in a 100 μl volume.
• Purpose: Detection of CD172a expression on myeloid cells, typically ex vivo.
• Example: Bone marrow cells from C57BL/6 mice stained with CD11b APC and P84 (purified antibody).

Bulk In Vivo Antibody Supply

• Available Concentration: 1.0–5.0 mg/ml.
• Formulation: 0.01 M PBS, pH 7.2, no carrier protein or preservatives.
• Endotoxin: ≤0.75 EU/mg, suitable for in vivo use.
• Administration: Actual dose administered to mice not specified in the product datasheet, but packaging is optimized for in vivo experimentation.

Additional Notes

• General In Vivo Use: While some vendors (e.g., Leinco, ichorbio) offer P84 specifically formulated for in vivo use, they do not specify a one-size-fits-all dosing regimen, instead highlighting purity, low endotoxin, and absence of carrier proteins or preservatives as key features.
• Flow Cytometry: For cell-surface staining, a concentration of ≤1.0 μg per 10⁶ cells is standard.
• Dose Reporting: The 20 μg/mouse/injection regimen in the NXS2 model is one of the few clearly documented in vivo dosing examples from the peer-reviewed literature.
• Infection Model: A MedchemExpress datasheet references a dose of 100 μg in Salmonella typhimurium infectious mouse models (Nr2f6-deficient mice), but this is not corroborated by primary literature in the search results and should be validated in peer-reviewed contexts.

Summary Table

Conclusion

Dosing regimens for clone P84 vary considerably across mouse models, with published in vivo regimens typically around 20 μg per mouse per injection for therapeutic studies, while flow cytometry uses much smaller amounts per cell. The dosing should be tailored to the specific experimental endpoint, and researchers are encouraged to consult both published literature and antibody provider protocols for optimal use. For new or specialized models, dose-finding studies may still be necessary.