Recombinant Mouse CD86 (B7-2)

Recombinant Mouse CD86 is widely used in research because it is a key costimulatory molecule involved in T cell activation, immune regulation, and serves as a valuable tool for studying immune responses, cell signaling, and therapeutic targeting in mouse models.

Key reasons to use recombinant mouse CD86 in research applications:

• T Cell Activation and Costimulation: CD86 (also known as B7-2) is essential for providing the costimulatory signal required for T-lymphocyte proliferation and interleukin-2 production by binding to CD28 or CTLA-4 on T cells. This interaction is fundamental for initiating and regulating immune responses, making recombinant CD86 a critical reagent for in vitro T cell activation assays and immunological studies.

• Functional Assays: Recombinant mouse CD86 can be immobilized and used to study its binding to ligands such as CTLA-4 and CD28, enabling quantitative analysis of these interactions. This is important for dissecting the molecular mechanisms of immune checkpoint regulation and for screening potential modulators of T cell activation.

• Immunotherapy and Biomarker Research: CD86 is implicated as a prognostic biomarker and potential therapeutic target in various diseases, including cancer. Recombinant CD86 allows for the development and validation of diagnostic assays, as well as the exploration of new immunotherapeutic strategies.

• Regulatory T Cell (Treg) Studies: CD86 is the dominant ligand for CD28-mediated proliferation and maintenance of FoxP3+ regulatory T cells, which are crucial for immune tolerance and homeostasis. Recombinant CD86 is thus valuable for investigating Treg biology and immune regulation.

• Antigen Presentation and Dendritic Cell Maturation: CD86 expression is rapidly induced on antigen-presenting cells (APCs) such as dendritic cells during immune responses, and its presence is used as a marker for APC activation and maturation. Recombinant CD86 can be used to stimulate or block these pathways in vitro.

• High Purity and Consistency: Recombinant proteins provide high purity and batch-to-batch consistency, which is essential for reproducible experimental results and for applications requiring defined molecular interactions.

• Versatility in Experimental Design: Recombinant mouse CD86 can be engineered with various tags (e.g., His, Fc) for easy detection, purification, or immobilization, and can be used in ELISA, flow cytometry, cell culture, and binding assays.

In summary, using recombinant mouse CD86 enables precise, reproducible, and mechanistic studies of immune costimulation, checkpoint regulation, and therapeutic targeting in mouse models, supporting a wide range of immunological and translational research applications.

Yes, recombinant Mouse CD86 can be used as a standard for quantification or calibration in ELISA assays, provided it is purified and its concentration is accurately determined. The standard curve in an ELISA requires a known concentration of the target protein, and recombinant proteins are commonly used for this purpose.

Key considerations:

• The recombinant protein should be highly purified and free of contaminants that could interfere with the assay.
• The concentration of the recombinant protein must be measured precisely, typically using methods such as amino acid analysis, spectrophotometry, or HPLC.
• The recombinant protein should be compatible with the detection antibodies used in your ELISA kit to ensure accurate quantification.

Several sources confirm that recombinant Mouse CD86 is suitable for use as a standard in ELISA assays, including OriGene (EA102207), BioLegend, and Innovative Research, which provide lyophilized recombinant Mouse CD86 specifically for ELISA standard use. Always refer to the manufacturer's instructions for the specific recombinant protein and ELISA kit to ensure compatibility and optimal performance.

Recombinant mouse CD86 protein has been validated for several applications in published research, primarily focusing on its functional and biochemical roles in immunology. Based on the available literature and product data, the main applications include:

• Functional Studies (Bioassays): Recombinant mouse CD86 is used to study its interaction with receptors such as CD28 and CTLA-4. For example, it has been immobilized to assess binding to CTLA-4 (both mouse and rat), demonstrating its ability to mediate costimulatory and inhibitory signals in T cell activation and regulation.

• ELISA: The protein is suitable as a capture or detection reagent in ELISA assays to quantify CD86 or its binding partners.

• SDS-PAGE: Used for purity and molecular weight analysis, with the protein migrating at 40–55 kDa due to glycosylation.

• Cell Culture and Immune Modulation Studies: Recombinant CD86 is employed in studies investigating T cell activation, costimulation, and immune regulation, including the modulation of T cell responses and cytokine production.

• Immunotherapy Research: CD86 has been studied as a therapeutic target in diseases such as classical Hodgkin lymphoma (cHL), where its blockade or targeting has shown efficacy in reversing T cell exhaustion and enhancing anti-tumor immunity.

• Flow Cytometry and Immunohistochemistry: While these applications are more commonly associated with CD86 antibodies, recombinant CD86 is sometimes used as a reference or control in these assays.

These applications highlight the utility of recombinant mouse CD86 in both basic research and translational immunology studies.

To reconstitute and prepare Recombinant Mouse CD86 protein for cell culture experiments, follow these general guidelines based on standard protocols and product specifications:

Reconstitution

1. Centrifuge the vial before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein with sterile, distilled water. The recommended concentration is typically 0.1–0.5 mg/mL (100–500 µg/mL), but some suppliers suggest up to 400 µg/mL. Always check the specific product datasheet for exact instructions.
3. Gently mix the solution by swirling or pipetting up and down. Avoid vigorous shaking to prevent foaming and protein denaturation.
4. Allow the vial to reconstitute for 15–30 minutes at room temperature with gentle agitation.

Storage

• Short-term storage: Reconstituted protein can be stored at 4–8°C for 2–7 days.
• Long-term storage: Aliquot the reconstituted protein and store at –20°C to –70°C. Avoid repeated freeze-thaw cycles.

Preparation for Cell Culture

1. Dilute the reconstituted protein to the desired concentration using serum-free medium or PBS. For coating plates, typical concentrations range from 1–10 µg/mL.
2. Add the diluted protein to the culture surface (e.g., wells of a plate).
3. Incubate at room temperature for 1–2 hours to allow the protein to adhere to the surface.
4. Aspirate the remaining protein solution and rinse the plate carefully with distilled water to remove unbound protein.
5. Plates are now ready for use. They can be stored at 2–8°C if kept damp or air-dried, provided sterility is maintained.

Additional Tips

• Always use sterile techniques to prevent contamination.
• Follow the specific instructions provided by the supplier, as protocols may vary slightly between products.
• For functional studies, ensure the protein is biologically active and has low endotoxin levels (<1 EU/µg).

By following these steps, you can effectively reconstitute and prepare Recombinant Mouse CD86 protein for use in cell culture experiments.