Recombinant Human IL-9

Recombinant Human IL-9 is used in research applications to study its diverse roles in immune regulation, hematopoiesis, inflammation, and cancer immunotherapy. It is a potent cytokine that modulates the function and proliferation of various immune and non-immune cells, making it valuable for both basic and translational research.

Key scientific applications and rationale for using recombinant human IL-9 include:

• Immune Cell Modulation: IL-9 acts on multiple hematopoietic cells, including T cells, B cells, mast cells, and eosinophils. It promotes the expansion and recruitment of mast cells and eosinophils, enhances IgE and Th2 cytokine production, and supports B cell antibody responses.
• T Cell Function and Anti-Tumor Immunity: IL-9 can enhance the cytolytic activity and survival of cytotoxic CD8+ T cells, contributing to anti-tumor responses. It has been shown to improve the efficacy of adoptive cell therapies by promoting a unique T cell phenotype with both stem cell memory and effector functions, leading to superior tumor infiltration and cytotoxicity in preclinical models.
• Hematopoietic Cell Proliferation: Recombinant IL-9 stimulates the proliferation of certain hematopoietic cell lines (e.g., MO7e, megakaryoblastic leukemia cells) and synergizes with other growth factors (such as Steel Factor) to enhance progenitor cell expansion.
• Inflammation and Allergy Models: IL-9 is implicated in allergic inflammation and asthma, as it can exacerbate mast cell-dependent responses and promote eosinophil development. Recombinant IL-9 is used to model and dissect these pathways in vitro and in vivo.
• Cell Survival and Anti-Apoptotic Effects: IL-9 prevents apoptosis in target cells, supporting studies on cell survival mechanisms and tissue repair.
• Keratinocyte Biology: IL-9 influences keratinocyte proliferation, survival, and cytokine secretion, making it relevant for skin biology and wound healing research.

Best practices for using recombinant human IL-9:

• Use in defined, serum-free media to study direct cytokine effects.
• Titrate concentrations for dose-response studies, as effects can be cell type- and context-dependent.
• Combine with other cytokines or growth factors to investigate synergistic or antagonistic interactions.

In summary, recombinant human IL-9 is a versatile tool for dissecting cytokine signaling, immune regulation, hematopoiesis, and disease mechanisms, as well as for developing and optimizing immunotherapeutic strategies.

Yes, recombinant human IL-9 can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay system and the antibodies used in your kit.

Supporting details:

• Many commercial ELISA kits for human IL-9 explicitly state that their standard curve is generated using recombinant human IL-9, and that the assay can quantify both natural and recombinant forms of the cytokine.
• Recombinant IL-9 is commonly supplied as a lyophilized protein, and protocols typically recommend reconstituting it in PBS or another suitable buffer for use as a standard in sandwich ELISA formats.
• The use of recombinant IL-9 as a standard is validated in multiple kit instructions and datasheets, which detail its application for calibration and quantification in plasma, serum, and cell culture supernatant samples.
• It is important to ensure that the recombinant IL-9 standard is of high purity and properly reconstituted, and that it matches the detection specificity of the antibodies in your ELISA system. Some kits specify the use of carrier proteins (e.g., BSA) for stability, while carrier-free formulations are recommended if BSA may interfere with your assay.

Best practices:

• Always follow the manufacturer’s instructions for standard preparation, dilution, and storage to ensure accurate calibration.
• Confirm that the recombinant IL-9 standard is compatible with the capture and detection antibodies in your assay, as some kits specify particular antibody clones for optimal performance.
• Validate the standard curve in your specific assay context, especially if using a recombinant standard from a different source than the kit manufacturer.

Summary: Recombinant human IL-9 is widely accepted and routinely used as a standard for ELISA quantification and calibration, as long as it is compatible with your assay reagents and protocol.

Recombinant Human IL-9 has been validated in published research for a range of applications, primarily in bioassays, cell proliferation studies, immune cell activation, and functional assays involving hematopoietic and immune cells.

Key validated applications include:

• Cell Proliferation Assays: IL-9 induces proliferation of the MO7e human megakaryocytic leukemic cell line, a standard bioactivity assay for recombinant IL-9.
• T Cell Stimulation and Differentiation: Used to stimulate T cells, including Th9, Th17, Treg, and Th2 subsets, and to study their development and function.
• Mast Cell Activation: IL-9 enhances mast cell expansion and recruitment, and is used to study mast cell biology.
• Allergic Inflammation Models: Validated for investigating allergic responses, including asthma and contact dermatitis, by promoting IgE and Th2 cytokine production and recruiting eosinophils.
• Autoimmunity and Immune Regulation: Applied in studies of autoimmune inflammation and Treg-mediated immune suppression.
• Apoptosis and Cell Survival: Used to assess anti-apoptotic effects and cell survival, such as Jak3-dependent survival in lymphoma cells.
• Airway Epithelium Repair: Shown to induce goblet cell hyperplasia during airway epithelial repair.
• Functional Assays in PBMCs: Enhances IFN-γ secretion in peripheral blood mononuclear cells (PBMCs), relevant for immunological studies.
• Adoptive Cell Therapy Research: Engineered T cells with synthetic IL-9 receptors demonstrate improved anti-tumor activity in preclinical models.

Additional validated uses:

• Chemokine Production and Migration Assays: Studied for its role in airway smooth muscle migration and chemokine production.
• Parasite Clearance Models: Used in research on gastrointestinal parasite immunity.
• Gene Therapy and Cell Therapy Solutions: Utilized in cell culture and processing for bioprocessing and therapeutic development.

Sample types: Most studies use whole cells, including human cell lines, primary immune cells, and PBMCs.

Summary Table of Validated Applications

These applications are supported by peer-reviewed publications and are widely used in immunology, cell biology, and translational research.

To reconstitute and prepare Recombinant Human IL-9 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or sterile buffer at a concentration of 0.1–0.2 mg/mL. After reconstitution, further dilute the stock solution in cell culture medium as needed for your assay.

Step-by-step protocol:

1. Centrifuge the vial briefly (20–30 seconds in a microcentrifuge) before opening to ensure all protein is at the bottom and to maximize recovery.
2. Add sterile water or buffer (e.g., PBS, pH 7.4) to achieve a final concentration of 100–200 μg/mL (0.1–0.2 mg/mL).
3. Gently mix by pipetting up and down. Do not vortex, as vigorous mixing may denature the protein.
4. Optional: Add a carrier protein (e.g., 0.1%–1% BSA or HSA) if you plan to store the stock solution or if the working concentration is below 0.5 mg/mL, to prevent protein loss due to adsorption to tube walls.
5. Aliquot the solution to avoid repeated freeze/thaw cycles.
6. Storage:
   • Use immediately or store aliquots at -20°C to -80°C for up to 3 months.
   • For short-term use (up to 1 week), store at 2–8°C.
   • Avoid repeated freeze/thaw cycles to maintain bioactivity.

Preparation for cell culture:

• Dilute the reconstituted stock solution into your cell culture medium to the desired final concentration, typically in the ng/mL range (e.g., ED50 for MO7e cell proliferation is 0.1–2 ng/mL, depending on the batch).
• If using serum-containing medium, consider serum starvation for certain assays, as serum may mask cytokine responses.

Additional notes:

• Confirm protein presence and integrity by SDS-PAGE if needed.
• Always use sterile technique to avoid contamination.
• Check the specific activity and endotoxin level provided in the product datasheet to ensure suitability for your application.

Summary Table:

This protocol ensures optimal recovery, stability, and bioactivity of recombinant human IL-9 for cell culture experiments.