Recombinant Human IL-10

Recombinant Human IL-10 (rhIL-10) is widely used in research applications due to its potent anti-inflammatory and immunomodulatory properties, making it valuable for studying immune regulation, inflammatory diseases, transplantation, and cancer immunology.

Key scientific applications and rationale:

• Anti-inflammatory effects: rhIL-10 suppresses the production of pro-inflammatory cytokines (e.g., IL-1, IL-6, IL-8, TNF-α), inhibits monocyte and T cell activation, and upregulates IL-1 receptor antagonist, thereby mitigating excessive immune responses in models of autoimmune and inflammatory diseases.

• Immunosuppression and tolerance induction: rhIL-10 promotes regulatory T cell (Treg) function and can prolong graft survival in transplantation models by reducing rejection and synergizing with other immunosuppressants.

• Cancer immunology: IL-10 has a dual role in cancer. It can stimulate cytotoxic CD8+ T cell activity and enhance interferon gamma production, contributing to anti-tumor immunity, while also suppressing tumor-promoting inflammation. Modified forms (e.g., PEGylated IL-10) have shown promise in preclinical and early clinical studies for solid tumors.

• Autoimmune and inflammatory disease models: rhIL-10 has been tested in clinical and preclinical studies for conditions such as Crohn’s disease, rheumatoid arthritis, and psoriasis, showing mild-to-moderate therapeutic benefits and safety.

• Infectious disease research: IL-10 is crucial for regulating immune responses during acute infections, preventing tissue damage from excessive inflammation, and is implicated in chronic infection persistence.

Best practices for research use:

• Employ rhIL-10 in cell culture or animal models to dissect mechanisms of immune regulation, cytokine signaling, and tolerance.
• Use rhIL-10 to modulate inflammatory responses in disease models or to test synergistic effects with other immunomodulatory agents.
• Consider pharmacokinetic limitations (short half-life) and potential need for modified forms (e.g., PEGylation) for sustained in vivo studies.

Summary of scientific value:Using recombinant human IL-10 enables precise control and investigation of anti-inflammatory pathways, immune tolerance, and cytokine interactions in diverse experimental systems, supporting translational research in immunology, transplantation, and oncology.

Yes, recombinant human IL-10 can be used as a standard for quantification or calibration in ELISA assays designed to measure human IL-10 concentrations.

Recombinant human IL-10 is widely accepted as a quantitative standard in ELISA protocols for human IL-10 detection. It is typically supplied at high purity (≥95%) and low endotoxin levels, making it suitable for generating standard curves in sandwich ELISA formats. The standard curve, constructed using serial dilutions of recombinant IL-10, allows for accurate quantification of IL-10 in biological samples such as serum, plasma, or cell culture supernatants.

Key technical considerations:

• Diluent consistency: Use the same calibrator diluent for both standards and samples to ensure accurate quantification.
• Carrier protein: For ELISA standards, carrier protein concentrations of 5–10 mg/mL are recommended to maintain stability and prevent loss of activity. Carrier proteins should be pre-screened for potential interference.
• Storage: Avoid diluting recombinant IL-10 below 50 μg/mL for long-term storage to preserve activity.
• Standard curve range: Typical standard curves use doubling dilutions from 2,000 pg/mL down to 15 pg/mL, but the exact range should match your assay’s sensitivity and expected sample concentrations.
• Parallelism: Standard curves generated with recombinant IL-10 are generally parallel to those obtained with natural human IL-10, supporting their use for relative mass quantification.

Best practices:

• Prepare a fresh standard curve for each assay run.
• Validate the recombinant standard in your specific assay system, especially if using custom antibodies or detection reagents.
• Follow manufacturer or published protocols for reconstitution, dilution, and storage of the recombinant IL-10 standard.

In summary, recombinant human IL-10 is a scientifically validated standard for ELISA quantification of human IL-10, provided that you follow recommended protocols for preparation, dilution, and assay setup.

Recombinant Human IL-10 has been validated for a broad range of applications in published research, primarily focused on its anti-inflammatory and immunomodulatory properties. The most commonly validated applications include:

• Bioassays: Used to assess IL-10’s biological activity in cell-based systems, including modulation of immune cell function, cytokine production, and signaling pathway activation (e.g., STAT1/STAT3 phosphorylation in monocytes and CD8⁺ T cells).
• Cell Culture: Applied to primary cells (such as monocytes, macrophages, B cells, T cells) and cell lines to study effects on proliferation, differentiation, cytokine secretion, and immune regulation.
• ELISA Standard: Utilized as a reference standard for quantifying IL-10 levels in biological samples via enzyme-linked immunosorbent assay.
• Blocking Control in Flow Cytometry: Used to block IL-10 activity or as a control in flow cytometric analysis, particularly when studying IL-10 receptor interactions.
• In Vivo Studies: Administered systemically in animal models (mouse, rat) to investigate therapeutic effects in autoimmune and inflammatory disease models.
• Clinical Trials: Tested in humans for treatment of autoimmune and immune-mediated inflammatory diseases such as inflammatory bowel disease (IBD), Crohn’s disease, rheumatoid arthritis, psoriasis, hepatitis C, and HIV.

Additional validated research applications include:

• Functional Assays: Investigation of IL-10’s role in suppressing proinflammatory cytokine production (e.g., IL-1α, IL-6, IL-8, TNF-α) and promoting regulatory T cell differentiation.
• Multiplexed Immunoassays: Used in screening for antibody specificity and immune profiling.
• Stem Cell Differentiation and Pluripotency Maintenance: Applied in protocols for stem cell culture and differentiation studies.
• Mass Spectrometry and HPLC: Characterization and quality control of recombinant IL-10 protein.

Key validated research areas:

• Modulation of innate and adaptive immune responses (e.g., inhibition of Th17 cells, promotion of regulatory T cells).
• Investigation of anti-inflammatory mechanisms in monocytes, macrophages, and endothelial cells.
• Therapeutic evaluation in preclinical and clinical models of autoimmune and inflammatory diseases.

These applications are supported by numerous peer-reviewed studies and clinical trials, demonstrating the versatility of recombinant human IL-10 in immunology, cell biology, and translational research.

To reconstitute and prepare Recombinant Human IL-10 protein for cell culture experiments, follow these best-practice steps:

1. Equilibrate and Centrifuge the Vial

   • Allow the lyophilized IL-10 vial to reach room temperature before opening to prevent condensation.
   • Briefly centrifuge the vial (20–30 seconds) to collect all material at the bottom and cap.

2. Reconstitution Buffer

   • Use sterile PBS (pH 7.2–7.4) or sterile water as the primary solvent.
   • For enhanced stability and to minimize adsorption, add 0.1%–1% carrier protein (such as endotoxin-free human or bovine serum albumin, HSA/BSA) to the buffer.
   • Example: For 0.2 mg/mL, dissolve in sterile 1x PBS pH 7.4 with 0.1% HSA.

3. Reconstitution Procedure

   • Add the recommended volume of buffer to achieve the desired stock concentration (commonly 0.1–1.0 mg/mL).
   • Gently swirl or tap the vial to dissolve the protein. Do not vortex or mix vigorously to avoid denaturation.
   • Allow the solution to sit for several minutes at room temperature to ensure complete dissolution.

4. Aliquot and Storage

   • Once fully dissolved, aliquot the stock solution into small volumes to avoid repeated freeze-thaw cycles.
   • Store aliquots at ≤–20°C (preferably in a manual defrost freezer). For short-term use (up to 1 week), 4°C is acceptable.
   • Avoid storage in frost-free freezers and minimize freeze-thaw cycles, as these can denature the protein.

5. Preparation for Cell Culture

   • Dilute the stock solution to the desired working concentration using cell culture medium (e.g., RPMI or DMEM) supplemented with serum (e.g., FBS) or with additional carrier protein if serum-free.
   • Typical working concentrations for functional assays range from 0.2–20 ng/mL, but optimize based on your specific experimental requirements.

6. Quality Check (Optional)

   • Confirm protein presence and integrity by SDS-PAGE if needed.

Summary Table: Key Steps and Conditions

Critical Notes:

• Always consult the product-specific Certificate of Analysis (CoA) for any unique instructions.
• Use sterile technique throughout to prevent contamination.
• If solubility issues arise, incubate the reconstituted solution at 4°C overnight.

These steps ensure optimal activity and stability of recombinant human IL-10 for cell culture applications.