Recombinant Mouse IL-12

Recombinant Mouse IL-12 is widely used in research because it is a potent immunomodulatory cytokine that plays a central role in regulating both innate and adaptive immune responses, particularly by promoting Th1 differentiation and enhancing cytotoxic activity of NK and T cells.

Key scientific applications and rationale for using recombinant mouse IL-12 include:

• Induction of Th1 Responses: IL-12 is essential for the differentiation of naïve CD4+ T cells into Th1 cells, which are critical for cell-mediated immunity against intracellular pathogens and tumors.
• Activation of NK and T Cells: It stimulates proliferation and activation of natural killer (NK) cells and cytotoxic T lymphocytes, leading to increased production of interferon-gamma (IFN-γ), a key cytokine in anti-tumor and anti-viral responses.
• Cancer Immunotherapy Research: Recombinant IL-12 has demonstrated strong anti-tumor effects in preclinical mouse models, reducing tumor growth and enhancing survival, especially when combined with other immunotherapies. It can induce long-term antitumor immunity and overcome tumor-associated immune suppression.
• Vaccine Adjuvant Studies: Due to its ability to promote cellular immunity, IL-12 is investigated as an adjuvant to enhance the efficacy of vaccines, particularly those requiring robust T cell responses.
• Functional Assays and Cell Culture: Recombinant mouse IL-12 is used in vitro to activate and expand NK and T cells, and to study cytokine signaling, immune cell differentiation, and effector functions.
• Modeling Immune Regulation: IL-12 is a valuable tool for dissecting pathways of immune regulation, autoimmunity, and inflammation in mouse models, providing insights into cytokine networks and immune cell interactions.

Summary of best practices:

• Use recombinant mouse IL-12 in controlled in vitro assays to study immune cell activation, differentiation, and cytokine production.
• Employ in vivo mouse models to investigate anti-tumor immunity, vaccine responses, or immune regulation, ensuring appropriate dosing and delivery methods to minimize toxicity and maximize efficacy.
• Combine with other cytokines or checkpoint inhibitors to explore synergistic effects in immunotherapy research.

In summary, recombinant mouse IL-12 is a powerful tool for immunological research, particularly in studies of Th1 immunity, cancer immunotherapy, and vaccine development, due to its well-characterized ability to drive potent cellular immune responses.

Yes, recombinant Mouse IL-12 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly formulated and validated for this purpose. Recombinant IL-12 is commonly used as a standard in quantitative sandwich ELISA kits designed to measure mouse IL-12 p70 in biological samples.

Key considerations and supporting details:

• Calibration and Standard Curve: ELISA kits for mouse IL-12 p70 are typically calibrated against highly purified recombinant mouse IL-12 p70. The standard curve is generated using serial dilutions of the recombinant protein, allowing quantification of IL-12 in unknown samples by comparison to this curve.

• Parallelism and Accuracy: Studies and kit documentation indicate that recombinant mouse IL-12 produces linear curves that are parallel to those obtained with natural mouse IL-12, supporting its use for relative mass quantification in ELISA assays.

• Formulation: For use as an ELISA standard, recombinant proteins are often supplied with carrier proteins such as BSA to enhance stability and prevent adsorption to plasticware. Carrier-free formulations are recommended if BSA or other carriers may interfere with your assay.

• Validation: It is essential to confirm that the recombinant IL-12 standard is recognized by the antibodies used in your ELISA and that it behaves similarly to endogenous IL-12 in your sample matrix. Most commercial ELISA kits specify compatibility with both natural and recombinant mouse IL-12 p70.

• No International Standard: Note that there is currently no international standard for mouse IL-12 p70 calibration, so quantification is relative to the recombinant standard used in your assay.

• Research Use Only: Recombinant standards and ELISA kits are intended for research use and not for diagnostic procedures.

Best Practices:

• Prepare the standard curve using the recombinant IL-12 according to the kit or assay protocol.
• Ensure the recombinant standard is stored and handled as recommended to maintain activity and stability.
• Validate recovery and parallelism in your specific sample matrix if using custom or non-kit ELISA formats.

In summary: Recombinant Mouse IL-12 is widely accepted and scientifically appropriate as a standard for ELISA quantification, provided it is properly formulated and validated for your assay system.

Recombinant Mouse IL-12 has been validated in published research for a range of immunological and cell biology applications, primarily involving the modulation and assessment of immune cell function.

Key validated applications include:

• In vitro activation and proliferation of NK and T cells: IL-12 is widely used to stimulate the growth and cytotoxic activity of natural killer (NK) cells and T lymphocytes in culture, supporting studies of immune cell function and differentiation.
• Differentiation of naïve CD4+ T cells toward Th1 lineage: IL-12 is a critical cytokine for driving Th1 polarization, making it essential in studies of T helper cell differentiation and immune response profiling.
• Stimulation of cytokine production: It promotes the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) by T cells and NK cells, which is frequently measured in functional assays and bioassays.
• Bioassays for immune modulation: IL-12 is used in bioassays to assess its effects on immune cell activation, cytokine secretion, and cytotoxicity, often as a positive control or to model immune responses in vitro and ex vivo.
• Anti-tumor and anti-viral research: Recombinant mouse IL-12 has been validated in preclinical models for its ability to enhance anti-tumor immunity, inhibit angiogenesis, and serve as a vaccine adjuvant by promoting cellular immunity.
• Functional studies in autoimmune and inflammatory disease models: IL-12 is used to modulate immune responses in models of autoimmune diseases, such as experimental autoimmune encephalomyelitis and type 1 diabetes, to study Th1/Th17 balance and inflammation.
• Protein-protein interaction and binding assays: It is employed in binding assays to study receptor interactions and downstream signaling pathways.
• Cell proliferation and cytotoxicity assays: IL-12 is used to assess its effects on immune cell proliferation and cytotoxic function, often in combination with other cytokines or stimuli.
• Mass spectrometry, SDS-PAGE, and HPLC: For protein characterization and quality control in research settings.

Representative published research applications:

• Enhancement of anti-tumor effects in cancer immunotherapy models.
• Investigation of T cell metabolism and stemness in adoptive cell transfer studies.
• Modulation of Th1/Th17 responses in autoimmune disease models.
• Studies of cytokine signaling and gene expression in T and NK cells.

These applications are supported by both product validation data and peer-reviewed publications, demonstrating the broad utility of recombinant mouse IL-12 in immunological research and preclinical studies.

To reconstitute and prepare Recombinant Mouse IL-12 protein for cell culture experiments, dissolve the lyophilized protein in sterile, pyrogen-free water or sterile PBS at a concentration of 100 μg/mL. After reconstitution, further dilute the stock solution in cell culture medium or buffer as required for your assay.

Step-by-step protocol:

• Reconstitution:

  • Add sterile distilled pyrogen-free water or sterile PBS (pH 6–8) to the lyophilized protein to achieve a final concentration of 100 μg/mL.
  • If recommended, include at least 0.1% carrier protein (such as human or bovine serum albumin) in PBS to stabilize the protein and prevent adsorption to tube walls.
  • Mix gently by pipetting up and down; avoid vigorous vortexing to prevent protein denaturation.
  • Allow the solution to sit at room temperature for 5–10 minutes to ensure complete dissolution.

• Aliquoting and Storage:

  • Aliquot the stock solution into small volumes to avoid repeated freeze-thaw cycles, which can reduce bioactivity.
  • Store aliquots at –20°C or colder for long-term storage. After reconstitution, the protein is stable for up to 1 month at 2–8°C under sterile conditions, or up to 3 months at ≤–20°C.
  • Avoid repeated freeze/thaw cycles.

• Preparation for Cell Culture:

  • Dilute the stock solution into your cell culture medium or buffer to the desired working concentration. Typical bioactive concentrations for mouse IL-12 in cell assays range from 0.01–0.1 ng/mL for stimulation of splenocytes or induction of IFNγ production.
  • Use sterile polypropylene tubes for all dilutions to minimize protein loss.

Additional Notes:

• If your application is sensitive to endotoxin, confirm the endotoxin level is suitable for cell culture.
• Always use sterile technique to prevent contamination.
• For optimal results, consult the specific activity (ED50) of your batch to determine the precise working concentration for your cell type and assay.

Summary Table:

This protocol ensures maximum bioactivity and stability of recombinant mouse IL-12 for cell culture experiments.