Recombinant Mouse B7-H2 (ICOSL)

Recombinant mouse B7-H2 (ICOSL) is a valuable tool for immunological research due to its critical role in T cell costimulation and its well-characterized functional properties.

Functional Significance

B7-H2 serves as a ligand for the inducible costimulator (ICOS), a CD28 family receptor expressed on activated T cells. When B7-H2 binds to ICOS on T cells, it delivers a potent costimulatory signal that enhances T cell proliferation and cytokine production. This interaction is distinct from the classical B7-1/B7-2-CD28 pathway, as B7-H2 uses overlapping but differential binding domains to interact with ICOS versus CD28 and CTLA-4.

Research Applications

The recombinant mouse B7-H2 protein enables several important experimental approaches:

T Cell Activation Studies: The protein can be used to investigate T cell proliferation and activation in response to costimulatory signals. When immobilized or presented as an Fc chimera, recombinant B7-H2 stimulates anti-CD3-induced T cell proliferation in a dose-dependent manner.

Cytokine Production Analysis: B7-H2 costimulation preferentially induces specific cytokine profiles compared to B7-1. While B7-1 generates high amounts of IL-2, B7-H2 appears to induce more IL-17A and can enhance IL-10 production through ICOS engagement. This makes it useful for studying Th1, Th2, and regulatory T cell responses.

Survival Signal Investigation: B7-H2-CD28 interaction provides crucial signals for enhancing Bcl-xL expression in activated CD4+ T cells, making it valuable for studying T cell survival mechanisms.

Immune Checkpoint Research: Since ICOS/ICOS-L signaling plays roles in both anti-tumor and pro-tumor immune responses, recombinant B7-H2 is applicable for investigating immune checkpoint biology and potential therapeutic combinations.

Technical Advantages

The recombinant protein typically exhibits high purity (>95% by SDS-PAGE) and maintains bioactivity suitable for functional assays. It is available in various formats, including Fc chimera constructs that facilitate immobilization and presentation to T cells. The lyophilized formulation provides stability for extended storage at -20°C to -70°C.

Yes, recombinant mouse B7-H2 (ICOSL) can be used as a standard for quantification and calibration in ELISA assays. This protein is specifically designed and validated for this application across multiple experimental platforms.

Primary Applications as an ELISA Standard

Recombinant mouse B7-H2 serves as a calibration standard to establish standard curves for quantifying endogenous B7-H2 levels in biological samples. The protein enables accurate determination of sample concentrations by providing known reference points across a range of concentrations. For example, commercial ELISA kits utilizing this protein demonstrate assay ranges such as 39-2500 pg/mL, allowing researchers to quantify B7-H2 in their samples with defined sensitivity parameters.

Formulation Considerations

When selecting recombinant B7-H2 for ELISA standardization, consider the formulation type: Proteins supplied with carrier proteins (such as bovine serum albumin) are recommended for use in cell or tissue culture applications and as ELISA standards. In contrast, carrier-free formulations are available for alternative applications but may require different handling considerations.

Important Limitation

A critical caveat exists regarding ELISA standard-grade recombinant proteins: they are not recommended for bioassay applications and have not been validated for functional assays. If your research requires both quantification via ELISA and functional characterization of B7-H2 activity, you should verify that your specific protein preparation has been tested for bioassay compatibility, as ELISA standard formulations are optimized for quantification purposes only.

For optimal results, ensure your standard curve preparation follows established protocols with appropriate dilution buffers and includes sufficient sample replicates across the dynamic range of your assay.

Recombinant Mouse B7-H2 (ICOSL) has been validated in published research for several key applications, primarily in studies of T cell costimulation, immune modulation, and protein-protein interaction assays.

Validated Applications in Published Research:

• In vitro T cell costimulation assays: Recombinant Mouse B7-H2 (ICOSL) is widely used to study its role as a co-stimulatory ligand for ICOS on activated T cells, promoting T cell proliferation and cytokine secretion. Immobilized recombinant B7-H2 can induce dose-dependent binding and activation of ICOS-expressing T cells.

• Protein-protein interaction studies: The recombinant protein is used in binding assays to characterize the interaction between B7-H2 and ICOS, often employing Fc-chimeric formats for immobilization and detection.

• Blocking/neutralization assays: Recombinant B7-H2 is used to validate antibodies or small molecules that block the ICOS–ICOSL interaction, which is relevant for immunotherapy and mechanistic studies of immune checkpoint pathways.

• Cellular functional assays: Studies have used recombinant B7-H2 to stimulate B cells and dendritic cells, assess proliferation, differentiation, and cytokine production, and to investigate its role in regulatory T cell development and tumor microenvironment modulation.

• In vivo immunomodulation: Engineered or recombinant ICOSL proteins have been used in animal models to modulate immune responses, including attenuation of T cell activation and investigation of therapeutic potential in autoimmune disease and cancer.

• Wound healing and angiogenesis models: Recombinant ICOSL stimulation has been shown to accelerate cutaneous wound healing and promote angiogenesis in mouse models.

Common Experimental Formats:

• ELISA standards: Recombinant B7-H2 is used as a standard for quantifying ICOSL in biological samples.
• Flow cytometry: Used for ligand binding and blocking assays to characterize cell surface expression and interaction.
• Biochemical assays: SDS-PAGE and CE-SDS are used to confirm purity and molecular weight for experimental reproducibility.

Summary Table of Applications

Key Insights:

• Recombinant Mouse B7-H2 (ICOSL) is a versatile tool for immunological research, especially in dissecting T cell costimulatory pathways and developing immunomodulatory therapies.
• Its use spans both basic mechanistic studies and translational research, including animal models of disease and therapeutic protein engineering.

If you require protocols or specific experimental details for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Mouse B7-H2 (ICOSL) protein for cell culture experiments, dissolve the lyophilized protein in sterile water or PBS to a concentration suitable for your assay, typically between 0.1–1.0 mg/mL. Use aseptic technique and include carrier protein (e.g., 0.1% BSA) if required for stability in cell-based assays.

Step-by-step protocol:

• Preparation:

  • Briefly centrifuge the vial to collect the lyophilized powder at the bottom.
  • Confirm the recommended diluent in the product datasheet or Certificate of Analysis. Most recombinant B7-H2 proteins are reconstituted in sterile distilled water or PBS, pH 7.2–7.3, without calcium or magnesium.
  • If using for cell culture, add 0.1% carrier protein (e.g., BSA) to PBS to minimize adsorption and stabilize the protein.

• Reconstitution:

  • Add sterile water or PBS to achieve a final concentration between 0.1–1.0 mg/mL (e.g., for 100 µg, add 100 µL for 1 mg/mL or 1 mL for 0.1 mg/mL).
  • Gently pipette up and down or swirl to dissolve. Avoid vigorous mixing or vortexing, which may denature the protein.
  • Allow the solution to sit at room temperature for a few minutes to ensure complete dissolution.

• Aliquoting and Storage:

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
  • Store aliquots at 2–8°C for up to one month or at –20°C to –70°C for long-term storage.
  • Avoid repeated freeze-thaw cycles, as these can reduce protein activity.

• Application in Cell Culture:

  • Dilute the stock solution in cell culture medium immediately before use.
  • Typical working concentrations for functional assays range from 1–10 µg/mL, but optimal concentrations should be determined empirically for your specific experiment.

Additional notes:

• Confirm endotoxin levels are below 0.1 EU/µg for cell culture applications to avoid unwanted immune activation.
• Always consult the lot-specific datasheet for precise instructions, as formulations and recommended protocols may vary.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant Mouse B7-H2 (ICOSL) for cell culture experiments.