Recombinant Mouse ICOS

Recombinant Mouse ICOS is a valuable tool for immunological research because it enables precise investigation of the ICOS (Inducible T-cell COStimulator) pathway, which is critical for T cell activation, T/B cell interactions, germinal center formation, and antibody class switching.

Key reasons to use recombinant Mouse ICOS in research applications:

• Dissection of T Cell Co-stimulation: ICOS is a co-stimulatory receptor on activated T cells, essential for their full activation, proliferation, and differentiation. Recombinant ICOS allows you to study these processes in vitro and in vivo, clarifying the molecular mechanisms underlying T cell responses.

• Modeling T/B Cell Interactions: The ICOS-ICOSL (ICOS Ligand) interaction is crucial for T cell help to B cells, germinal center formation, and antibody class switching. Using recombinant ICOS, you can mimic or block this interaction to study its effects on humoral immunity and memory responses.

• Therapeutic Target Validation: ICOS is implicated in autoimmune diseases, cancer immunotherapy, transplantation tolerance, and inflammatory conditions. Recombinant Mouse ICOS can be used to screen and validate therapeutic antibodies, small molecules, or biologics targeting this pathway.

• Functional Assays: Recombinant ICOS proteins (including Fc chimeras) are used in binding assays, cell signaling studies, and as reagents to stimulate or inhibit ICOS signaling in primary cells or cell lines.

• Structural and Mechanistic Studies: Recombinant ICOS enables structural biology approaches (e.g., crystallography, binding kinetics) to elucidate the molecular details of ICOS/ICOSL interactions and antibody recognition.

• Wound Healing and Angiogenesis Research: ICOS-Fc fusion proteins have been shown to promote wound healing and angiogenesis in mouse models, supporting their use in studies of tissue repair and regeneration.

• CAR-T Cell Engineering: Incorporation of ICOS domains into CAR-T cells enhances their persistence and antitumor activity, making recombinant ICOS useful for developing and testing next-generation cell therapies.

In summary, recombinant Mouse ICOS is essential for mechanistic, translational, and therapeutic studies focused on T cell biology, immune regulation, and disease modeling, providing a controlled and reproducible reagent for diverse experimental systems.

You can use recombinant Mouse ICOS as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with your assay's antibodies and detection system.

Key considerations and best practices:

• Purity and Quantification: The recombinant protein should be highly purified, and its concentration must be precisely known, typically determined by absorbance at 280 nm or another validated method. Impurities or inaccurate quantification can compromise the standard curve and assay accuracy.

• Standard Curve Preparation: Prepare a serial dilution of the recombinant ICOS in the same buffer or matrix as your samples to generate a standard curve. This allows for accurate interpolation of sample concentrations.

• Protein Form and Epitope Accessibility: Ensure that the recombinant ICOS is in a form (e.g., full-length, Fc chimera, tag placement) that is recognized by the capture and detection antibodies in your ELISA. Differences in folding or post-translational modifications between recombinant and native ICOS can affect antibody binding and quantification accuracy.

• Carrier Proteins: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to enhance stability. For ELISA standards, carrier-free preparations are often preferred to avoid interference, unless your assay is validated for use with carrier-containing standards.

• Validation: It is recommended to validate the recombinant ICOS standard in your specific ELISA setup. This includes checking for parallelism between the standard curve and serially diluted native samples, and performing spike-and-recovery experiments to confirm accuracy in your sample matrix.

• Controls: Include appropriate controls, such as an endogenous positive control (native ICOS from mouse samples), to ensure that the recombinant standard behaves similarly to the native protein in your assay.

Limitations:

• If the recombinant ICOS differs structurally from the native protein (e.g., due to tags, truncations, or lack of glycosylation), this may affect antibody recognition and quantification accuracy.
• Not all recombinant proteins are validated for use as ELISA standards; check the product documentation or perform in-house validation.

Summary Table:

In summary, recombinant Mouse ICOS can be used as an ELISA standard if it is properly validated and meets the above criteria. Always confirm compatibility with your specific assay system.

Recombinant Mouse ICOS has been validated for several key applications in published research, primarily in immunology and cell biology:

• Immunomodulation in vivo and in vitro: Recombinant soluble ICOS (often as ICOS-Fc fusion protein) has been used to modulate immune responses, acting as both an antagonist of ICOS and agonist of ICOSL. This approach has been validated in mouse models of sepsis, where ICOS-Fc administration reduced cytokine storm, modulated cytokine release (e.g., IL-1β, IL-6, IL-10, TNF-α), and improved clinical outcomes.

• Cell migration and adhesion assays: ICOS-Fc has been shown to inhibit endothelial and tumor cell adhesion and migration in vitro, as well as modulate dendritic cell function and osteoclast differentiation.

• Flow cytometry and receptor-ligand blockade: Recombinant Mouse ICOS is validated for use in flow cytometry to detect ICOS expression on T cells and other immune cells, and for blocking ICOS-ICOSL interactions in functional assays.

• Cellular therapy and CAR-T cell engineering: ICOS costimulation has been validated in the generation of metabolically fit and therapeutically potent CAR Th17 cells for adoptive cell therapy in mouse tumor models. ICOS signaling was shown to enhance antitumor efficacy and persistence of CAR-T cells.

• ELISA standard and cell/tissue culture: Recombinant Mouse ICOS is recommended as a standard for ELISA assays and as a reagent in cell or tissue culture experiments to study ICOS-mediated signaling.

• Comparative immunology and preclinical therapeutic evaluation: The protein is used in mouse model studies to investigate ICOS function in immune regulation, cytokine production, and as a target for therapeutic intervention.

Summary Table: Validated Applications of Recombinant Mouse ICOS

These applications are supported by peer-reviewed studies and technical protocols, demonstrating the versatility of recombinant Mouse ICOS in both basic and translational immunology research.

To reconstitute and prepare Recombinant Mouse ICOS protein for cell culture experiments, dissolve the lyophilized protein in sterile, distilled water or sterile PBS, aiming for a final concentration between 0.1–1.0 mg/mL depending on your experimental needs. Always consult the product’s Certificate of Analysis (CoA) for specific instructions, as buffer requirements may vary by construct or tag.

Essential steps and considerations:

• Centrifuge the vial briefly to collect the lyophilized powder at the bottom before opening.
• Add sterile water or PBS: For most recombinant proteins, sterile distilled water is suitable, but some preparations (especially Fc chimeras or tagged proteins) may require PBS or a specific buffer (e.g., 20 mM Tris, 150 mM NaCl, pH 8.0).
• Typical concentration: Reconstitute to 0.1–1.0 mg/mL. For example, add 100–1000 µL to 100 µg of protein.
• Gentle mixing: Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation. Avoid vigorous shaking or vortexing to prevent denaturation or foaming.
• Carrier protein (optional): If the protein is to be used at low concentrations (<10 µg/mL), consider adding 0.1% BSA or HSA to stabilize and prevent adsorption to plasticware.
• Sterile filtration: If required for cell culture, filter the reconstituted solution through a 0.2 µm filter to ensure sterility.
• Aliquot and storage: Aliquot the solution to avoid repeated freeze-thaw cycles. Store at −80°C for long-term use or at 4°C for short-term (days to weeks).

Preparation for cell culture:

• Endotoxin testing: Confirm the protein is endotoxin-free or has endotoxin levels suitable for cell culture (<0.1 EU/µg is typical for sensitive assays).
• Dilution in culture medium: Dilute the reconstituted stock into your cell culture medium immediately before use. Avoid prolonged incubation at room temperature.
• Buffer compatibility: Ensure the buffer used for reconstitution is compatible with your cell culture system (e.g., avoid high salt or non-physiological pH).

Summary Table:

Always verify the specific requirements for your recombinant Mouse ICOS protein batch, as tags (e.g., Fc, His) and expression systems (e.g., HEK293) may influence buffer choice and handling.