Recombinant Mouse LIGHT

Using Recombinant Mouse LIGHT (TNFSF14) in research applications is valuable for studying immune modulation, tumor immunology, and lymphoid tissue organization due to its well-characterized roles in activating immune responses and its ability to engage specific receptors in a controlled, reproducible manner.

Key scientific reasons to use recombinant mouse LIGHT:

• Immune Activation and Modulation: Recombinant mouse LIGHT binds to the receptors HVEM (herpesvirus entry mediator, TNFRSF14) and LTβR (lymphotoxin beta receptor, TNFRSF3), both of which are critical for regulating innate and adaptive immune responses. Engagement of HVEM by LIGHT provides potent costimulatory signals to T cells and NK cells, promoting their differentiation into effector cells and enhancing cytotoxic activity.

• Tumor Immunology Applications: Soluble recombinant mouse LIGHT, especially in its trimeric form (e.g., Ig.Foldon-mLIGHT), has been shown to promote immune cell infiltration into tumors, delay tumor growth, and enhance anti-tumor immunity in mouse models. This effect is mediated by increased recruitment of dendritic cells and cytotoxic lymphocytes to the tumor microenvironment, making it a promising tool for preclinical cancer immunotherapy research.

• Lymphoid Tissue Organization: LIGHT-LTβR signaling is involved in the development and restoration of secondary lymphoid structures, which are essential for effective immune responses and tissue homeostasis. This makes recombinant LIGHT useful for studies on lymphoid organogenesis and tissue engineering.

• Cytokine and Chemokine Induction: LIGHT signaling induces the production of various cytokines and chemokines, facilitating leukocyte recruitment, endothelial activation, and modulation of the tumor microenvironment.

• Controlled and Reproducible Experimental Conditions: Recombinant proteins offer batch-to-batch consistency, defined activity, and high purity, which are critical for reproducible results in mechanistic studies and therapeutic development.

Additional applications and considerations:

• Apoptosis Induction: LIGHT can induce apoptosis in certain tumor cells, providing a direct anti-tumor mechanism for in vitro and in vivo studies.
• NF-κB Activation: LIGHT is a potent activator of the NF-κB pathway, a central regulator of inflammation and immune responses.
• Combination Therapy Research: Recombinant LIGHT can be used in combination with immune checkpoint inhibitors to investigate synergistic effects in cancer models.

Technical note: The biological activity of recombinant mouse LIGHT depends on its trimeric structure, which mimics the natural ligand configuration required for effective receptor engagement. Recent advances in protein engineering (e.g., foldon-mediated trimerization) have enabled the production of functionally active recombinant LIGHT suitable for in vivo and in vitro applications.

In summary, recombinant mouse LIGHT is a powerful tool for dissecting immune pathways, modeling tumor-immune interactions, and exploring therapeutic strategies in immunology and oncology research.

Recombinant Mouse LIGHT (TNFSF14) protein can be used as a standard for quantification or calibration in ELISA assays, provided it is specifically formulated and validated for this purpose.

Essential context and best practices:

• Formulation: Recombinant proteins intended for use as ELISA standards are typically supplied in carrier-containing formulations (e.g., with BSA) to enhance stability and reproducibility. Carrier-free formulations may be less stable and more prone to adsorption losses, which can affect quantification accuracy.

• Validation: Not all recombinant proteins are validated for use as ELISA standards. Proteins sold as "ELISA standards" have undergone testing to ensure their performance in standard curve generation and quantification. If using a recombinant LIGHT protein not specifically validated as an ELISA standard, you must perform your own validation, including spike/recovery and linearity experiments, to confirm its suitability.

• Calibration: Standards provided in commercial ELISA kits are calibrated against a master calibrator and are traceable for quantification. If you use a recombinant LIGHT protein from a separate source, you should calibrate it against a known mass standard or assign its value based on measurement in your ELISA system, rather than relying solely on the mass stated on the vial.

• Assay compatibility: Ensure that the recombinant LIGHT protein matches the epitope recognized by your ELISA antibodies (capture and detection), and that its biological activity or immunoreactivity is comparable to the native protein in your samples.

• Controls: Include appropriate controls, such as endogenous positive controls and negative controls, to validate quantification accuracy when using recombinant proteins as standards.

Summary of protocol recommendations:

• Use recombinant Mouse LIGHT protein formulated and validated for ELISA standard use.
• Perform spike/recovery and linearity validation if using a non-validated recombinant protein.
• Calibrate the recombinant standard in your assay system, not solely by vial mass.
• Confirm epitope compatibility and immunoreactivity with your ELISA antibodies.
• Include appropriate controls in your assay design.

Limitations:
If the recombinant Mouse LIGHT protein is not specifically validated for ELISA standard use, quantification accuracy may be compromised. Always validate the standard in your specific assay context before relying on it for quantitative measurements.

Recombinant Mouse LIGHT has been validated in published research primarily for applications in tumor immunology, immune cell activation assays, and mechanistic studies of receptor engagement.

Key validated applications include:

• In vivo tumor models: Recombinant mouse LIGHT (specifically, engineered soluble forms such as Ig.Foldon-mLIGHT) has been used in syngeneic mouse models (e.g., B16.F10 melanoma) to demonstrate its ability to delay tumor growth, promote immune cell infiltration (dendritic cells and cytotoxic lymphocytes), and modulate the tumor microenvironment. These studies validate its use in preclinical cancer immunotherapy research.

• In vitro receptor binding and signaling assays: The recombinant protein has been shown to bind its physiological receptors (HVEM and LTβR) and activate downstream signaling pathways, such as NF-κB activation, in cell-based assays. This validates its use in mechanistic studies of TNF superfamily signaling.

• Immune cell activation and proliferation assays: LIGHT has been validated for stimulating proliferation of lymphocytes, inducing apoptosis in certain tumor cells, and promoting cytokine production (e.g., IL-12 by dendritic cells, IFN-γ by CD4+ T cells) in vitro.

• Flow cytometry and ELISA: Recombinant LIGHT has been used as a standard or detection reagent in flow cytometry (to confirm cell surface expression) and sandwich ELISA (to quantify secreted protein in engineered cell lines).

• Functional studies in chronic inflammation models: LIGHT has been used in mouse models of chronic asthma to study its role in lung fibrosis, airway hyperresponsiveness, and immune modulation, validating its application in inflammation research.

Summary Table of Validated Applications

Additional notes:

• The most robust validation is in the context of tumor immunology, where recombinant mouse LIGHT has been shown to modulate the tumor microenvironment and enhance anti-tumor immunity.
• Functional trimerization (e.g., via foldon domains) is critical for activity in recombinant constructs.
• LIGHT’s effects are mediated through its receptors HVEM and LTβR, and these interactions are central to its validated applications.

No evidence was found in the provided results for use in diagnostic or clinical applications; all validations are in research contexts.

To reconstitute and prepare Recombinant Mouse LIGHT protein for cell culture experiments, dissolve the lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). This carrier protein helps stabilize LIGHT and prevents adsorption to plastic surfaces, which is critical for maintaining activity in cell-based assays.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile PBS (phosphate-buffered saline) with at least 0.1% BSA to the vial to reach a final concentration of 100 μg/mL.
• Gently mix by swirling or inverting; avoid vigorous shaking or vortexing to prevent protein denaturation and foaming.
• Allow the protein to fully dissolve at room temperature for 15–30 minutes with gentle agitation.
• Aliquot the reconstituted protein into working volumes to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage:
  • Short-term (up to 1 week): Store at 2–8 °C.
  • Long-term: Store aliquots at –20 °C or –80 °C. For extended storage, consider adding 5–50% glycerol as a cryoprotectant.

Preparation for cell culture:

• Before adding to cells, dilute the reconstituted LIGHT protein to the desired working concentration using cell culture medium or PBS with 0.1% BSA.
• Filter-sterilize if necessary, especially if sterility is critical for your application.
• Add the protein directly to the cell culture medium at the required final concentration for your experiment.

Additional notes:

• Always consult the specific product datasheet for any manufacturer-specific recommendations regarding buffer composition or reconstitution procedure, as some formulations may differ (e.g., some may recommend reconstitution in 100 mM acetic acid for certain lots).
• Avoid repeated freeze-thaw cycles by aliquoting immediately after reconstitution.
• For functional assays, confirm the activity of the reconstituted protein using a relevant bioassay if possible.

This protocol ensures optimal stability and biological activity of recombinant mouse LIGHT for cell culture applications.