Recombinant Mouse IL-2

Recombinant Mouse IL-2 is widely used in research because it is a critical cytokine for modulating immune responses, particularly in studies involving T cell biology, immune regulation, and immunotherapy models.

Key reasons to use recombinant mouse IL-2 in research applications:

• T Cell Growth and Activation: IL-2 is essential for the proliferation, differentiation, and survival of antigen-specific cytotoxic T cells, as well as for the activation and maintenance of immune responses. It is often used to stimulate T cell cultures in vitro and to expand T cells for functional assays.

• Regulatory T Cell (Treg) Expansion: IL-2 is crucial for the development, expansion, and maintenance of CD4⁺CD25⁺Foxp3⁺ regulatory T cells, which are central to immune tolerance and the prevention of autoimmunity. Low-dose IL-2 is specifically used to selectively expand Tregs in both in vitro and in vivo models.

• Natural Killer (NK) Cell Activation: IL-2 promotes the proliferation and activation of NK cells, making it valuable for studies on innate immunity and NK cell-mediated cytotoxicity.

• Immunotherapy and Cancer Research: IL-2 is a potent immunostimulant and has been used in preclinical and clinical studies for cancer immunotherapy, including the activation of effector T cells and NK cells to target tumors.

• Autoimmunity and Tolerance Studies: Recombinant IL-2 is used to model and manipulate immune tolerance, particularly in autoimmune disease models, by modulating the balance between effector T cells and Tregs.

• Standardization and Reproducibility: Recombinant IL-2 provides a consistent, defined, and animal-free source of cytokine, ensuring reproducibility and precision in cell culture, ELISA standards, and Western blot controls.

• Mechanistic Studies: IL-2 is used to dissect signaling pathways (e.g., JAK/STAT, PI3K/AKT, Ras/Raf/MAPK) involved in T cell activation and survival, as well as to study the molecular mechanisms underlying immune cell differentiation and function.

In summary, recombinant mouse IL-2 is indispensable for immunological research involving T cell and NK cell biology, immune regulation, cancer immunotherapy, and studies of immune tolerance and autoimmunity, due to its well-characterized and central role in immune modulation.

Yes, recombinant mouse IL-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly validated for your specific assay system.

Key considerations and supporting details:

• Intended Use: Recombinant mouse IL-2 is widely used as a quantitative standard in mouse IL-2 ELISA assays, especially in sandwich ELISA formats. It is suitable for generating standard curves to quantify IL-2 concentrations in experimental samples.

• Purity and Formulation: The recombinant IL-2 standard should be ≥95% pure, as determined by SDS-PAGE or similar methods, and free from significant endotoxin contamination. It is typically supplied lyophilized and should be reconstituted with a buffer containing a carrier protein (such as BSA) to prevent adsorption and loss of activity.

• Standard Curve Preparation: Prepare a series of dilutions (often doubling dilutions) of the recombinant IL-2 standard to generate a standard curve covering the expected range of your samples (e.g., 2,000 to 15 pg/mL or as specified by your assay protocol). The standard curve allows interpolation of sample absorbance values to determine IL-2 concentrations.

• Assay Compatibility: Ensure that the recombinant IL-2 standard is compatible with the capture and detection antibodies used in your ELISA. Some protocols specify particular antibody clones for optimal performance. If using a kit, follow the manufacturer's recommendations for standard preparation and curve generation.

• Calibration and Traceability: Some commercial ELISA kits calibrate their recombinant IL-2 standards against international reference materials (e.g., NIH/NIBSC standard lot 93/566), which can improve inter-assay comparability. If traceability is important for your application, check if your recombinant IL-2 standard is calibrated accordingly.

• Sample Matrix Considerations: While recombinant IL-2 is suitable as a standard for cell culture supernatants, some ELISA systems may require matrix-matched standards for serum or plasma samples due to potential matrix effects. Always validate recovery and parallelism if quantifying IL-2 in complex biological fluids.

• Validation: For quantitative accuracy, validate your standard curve by running it in parallel with known concentrations and checking for linearity and reproducibility. Intra- and inter-assay coefficients of variation (CV) should be <10% for reliable quantification.

Summary Table: Recombinant Mouse IL-2 as ELISA Standard

In summary: Recombinant mouse IL-2 is appropriate as a standard for ELISA quantification, provided it is of high quality, properly prepared, and validated for your assay system. Always follow best practices for standard preparation and assay validation to ensure accurate and reproducible results.

Recombinant Mouse IL-2 has been validated for a broad range of applications in published research, primarily in immunology and cell biology. The most common validated applications include:

• In vitro activation and expansion of T cells: Recombinant mouse IL-2 is widely used to stimulate and propagate mouse T cells in culture, supporting studies of T cell function, differentiation, and proliferation.
• Expansion and activation of NK cells: It is used to activate and expand natural killer (NK) cells for functional assays and cytotoxicity studies.
• Generation of lymphokine-activated killer (LAK) cells: IL-2 is a key factor in generating LAK cells for cytotoxicity assays and cancer immunotherapy research.
• Bioassays and functional assays: IL-2 is used as a standard or stimulant in various bioassays to assess cell proliferation, cytokine production, and immune cell activation.
• In vivo studies: Recombinant mouse IL-2 has been validated in animal models for:
  • Regulatory T cell (Treg) expansion and immune tolerance induction, including transplantation tolerance and autoimmune disease models.
  • Cancer immunotherapy: Used to enhance anti-tumor immune responses in mouse tumor models.
  • Assessment of immune modulation in infectious disease, autoimmunity, and vaccine studies.

Additional validated applications include:

• ELISA standard: Used as a quantitative standard in immunoassays for IL-2 detection.
• Cell migration and motility assays: To study the effects of IL-2 on immune cell movement.
• Cell differentiation studies: To investigate the role of IL-2 in the differentiation of various immune cell subsets.

Summary Table: Validated Applications of Recombinant Mouse IL-2

These applications are supported by both product validation data and numerous peer-reviewed studies, confirming the utility of recombinant mouse IL-2 in both basic and translational immunology research.

Reconstitution Protocol

Reconstituting recombinant mouse IL-2 requires careful attention to sterile technique and proper handling to maintain protein activity. The reconstitution concentration typically ranges from 100-200 μg/mL, though some protocols recommend 0.1-1.0 mg/mL depending on your specific application.

Initial Preparation Steps

Begin by removing the lyophilized vial from the -80°C freezer and allowing it to warm to room temperature naturally without artificial heat, which takes approximately 1 hour. Once warmed, spray the vial with 70% ethanol and place it in a laminar flow hood to maintain sterile conditions. Before opening, briefly centrifuge the vial to bring the contents to the bottom.

Reconstitution Solutions

The choice of reconstitution buffer depends on the specific formulation:

• With carrier protein formulation: Reconstitute in sterile phosphate-buffered saline (PBS) containing at least 0.1% human or bovine serum albumin (BSA)
• Carrier-free formulation: Reconstitute in sterile deionized water or high-purity water (18 MΩ-cm)
• Alternative protocol: Some preparations use 100 mM acetic acid for reconstitution

Reconstitution Procedure

Add the appropriate buffer slowly to minimize splashing and overflow. For example, add 5.2 mL of sterile PBS to reconstitute to approximately 100,000 U/mL. Gently mix the vial to ensure complete dissolution of the lyophilized powder—do not vortex, as this can denature the protein. Allow the solution to sit for approximately 10 minutes to ensure complete reconstitution.

If using an acetic acid-based protocol, filter the reconstituted solution through a sterile 0.45 micron filter into a sterile collection tube.

Storage and Stability

Long-term Storage

Store reconstituted aliquots at -80°C for long-term stability. Use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can significantly reduce protein activity.

Short-term Storage

Reconstituted protein can be maintained at -20°C for 1-2 weeks. At 2-8°C under sterile conditions, the protein remains stable for up to 1 month. Aliquots stored at 2-8°C can be used for up to one week.

Aliquoting for Cell Culture Use

Divide the reconstituted stock into working aliquots of appropriate volumes (typically 200 μL per tube) in sterile 1.7 mL screw-cap tubes. Label each tube with the concentration, date, and storage temperature. This approach minimizes the need to thaw the entire stock repeatedly and maintains protein integrity throughout your experiments.

When preparing working solutions for cell culture, further dilute the stock in low endotoxin medium or buffered solutions containing fetal bovine serum (FBS) or tissue culture-grade BSA. For optimal bioactivity in stimulation assays, the effective concentration typically ranges from 0.1-0.4 ng/mL.