Recombinant Human IL-5

Recombinant Human IL-5 is widely used in research applications to study and manipulate the biology of eosinophils, B cells, and basophils due to its well-defined activity, high purity, and reproducibility.

Key scientific reasons to use Recombinant Human IL-5:

• Eosinophil Biology: IL-5 is the principal cytokine regulating eosinophil differentiation, maturation, activation, migration, and survival. Recombinant human IL-5 reliably induces these processes in vitro, making it essential for studies on eosinophil function, allergic inflammation, asthma, and hypereosinophilic syndromes.

• B Cell Activation: IL-5 can stimulate activated B cells to differentiate into antibody-secreting cells, supporting research into humoral immunity and antibody production.

• Basophil Priming: IL-5 also promotes basophil differentiation and primes them for mediator release, relevant for allergy and hypersensitivity research.

• Bioassays and Standards: Recombinant human IL-5 is used as a quantitative standard in ELISA assays for measuring IL-5 levels in biological samples, ensuring consistency and accuracy in cytokine quantification.

• Cell Proliferation Assays: It stimulates proliferation of indicator cell lines such as TF-1, providing a robust readout for cytokine activity and downstream signaling studies.

• Mechanistic Studies: Recombinant IL-5 enables controlled investigation of signaling pathways (e.g., JAK/STAT, Btk, Ras/ERK) activated by IL-5 receptor engagement.

Advantages of recombinant over native or tissue-derived IL-5:

• Purity and Consistency: Recombinant IL-5 is highly purified and batch-to-batch consistent, minimizing experimental variability and contamination risks.
• Scalability and Safety: Recombinant production allows for scalable supply and eliminates risks associated with animal-derived proteins, such as immunogenicity or pathogen transmission.
• Defined Activity: Recombinant IL-5 has a well-characterized specific activity (e.g., ED50 values for cell proliferation), enabling precise dosing and reproducible results.

Typical applications include:

• In vitro differentiation and activation of human eosinophils and B cells.
• Functional assays for cytokine signaling and immune cell responses.
• ELISA standards for cytokine quantification.
• Mechanistic studies of allergic inflammation, asthma, and immune regulation.

Using recombinant human IL-5 ensures experimental reliability and relevance for human immunology research, especially when studying eosinophil-mediated processes and cytokine signaling pathways.

Yes, recombinant human IL-5 is widely used as a standard for quantification or calibration in ELISA assays. This practice is well-established in the literature and by multiple ELISA kit protocols.

Key points and best practices:

• Recombinant human IL-5 is specifically recommended as a quantitative standard for measuring human IL-5 protein levels in sandwich ELISA formats.
• ELISA kits and protocols routinely use recombinant IL-5 to generate standard curves, allowing for the quantification of IL-5 in biological samples such as serum, plasma, cell culture supernatant, and urine.
• The standard curve should be freshly prepared for each assay plate to ensure accuracy, as technical errors in standard preparation can compromise quantification.
• Purity and formulation: Recombinant IL-5 used as a standard is typically ≥95% pure, with low endotoxin levels, and may be supplied with or without carrier protein. If using a carrier-free preparation, it is recommended to add a carrier protein (e.g., BSA) at 5–10 mg/mL to prevent loss of activity and adsorption to plasticware.
• Dilution range: Standard curves are commonly prepared using serial (often doubling) dilutions, covering a range such as 2,000–15 pg/mL or as specified by your assay’s sensitivity and dynamic range.
• Storage: For long-term storage, do not dilute below 1 μg/mL, and store at recommended temperatures (typically –20°C or –80°C). Avoid repeated freeze-thaw cycles.
• Matrix effects: When quantifying IL-5 in complex matrices (e.g., serum, plasma), ensure that the standard is diluted in a matrix-matched buffer or assay diluent to minimize matrix effects and improve accuracy.

Summary Table: Use of Recombinant Human IL-5 as ELISA Standard

Conclusion:
You can use recombinant human IL-5 as a standard for ELISA quantification, provided you follow best practices for preparation, dilution, and storage, and ensure compatibility with your assay system. Always consult your specific ELISA protocol for any additional requirements.

Recombinant Human IL-5 has been validated in published research for a range of applications primarily focused on eosinophil biology, immune cell signaling, and cytokine bioassays.

Key validated applications include:

• Bioassays: Used to stimulate cell proliferation, especially in the TF-1 human erythroleukemic cell line and human eosinophils. These assays assess IL-5’s biological activity, such as promoting eosinophil differentiation, survival, and activation.
• Chemotaxis and Migration Assays: Demonstrated as a potent and selective chemoattractant for human eosinophils, facilitating studies of eosinophil recruitment and migration.
• Cell Differentiation Studies: Validated for inducing eosinophil differentiation from progenitor cells and supporting maturation and survival of eosinophils in vitro.
• Immunological Functional Assays: Used to study cytokine signaling pathways, such as STAT5 and MAPK, and to investigate the regulation of apoptosis and cell survival in eosinophils.
• Ligand Blocking Controls: Applied as a specificity control in immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry to validate antibody binding and cytokine detection.
• ELISA and ELISPOT: Utilized as a standard or control in cytokine quantification assays, including ELISA and ELISPOT, for measuring IL-5 levels in biological samples.
• Western Blot and Immunoprecipitation: Used as a positive control or for validating antibody specificity in protein detection assays.
• Cell Culture Supplementation: Added to culture media to support eosinophil growth and differentiation in hematopoietic and immune cell studies.

Representative published research applications:

• Investigation of eosinophil-mediated inflammation and fibrosis in colitis models.
• Studies on IL-5’s suppression of VEGF-induced angiogenesis via STAT5 signaling.
• Analysis of eosinophil apoptosis and survival mechanisms in asthma and allergy models.
• Chemotaxis assays demonstrating selective migration of eosinophils in response to IL-5 gradients.
• Functional studies on B cell activation and differentiation, although human IL-5 shows limited activity compared to murine IL-5 in B cell growth assays.

Summary Table of Validated Applications

These applications are well-supported in peer-reviewed literature and product citations, making recombinant human IL-5 a standard tool for immunology, hematopoiesis, and allergy research.

To reconstitute and prepare Recombinant Human IL-5 protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Reconstitute the protein in sterile water or PBS:
  • Typical concentrations for reconstitution are 0.1–1.0 mg/mL in sterile distilled water or PBS.
  • Some protocols recommend at least 50 μg/mL in PBS containing 0.1% BSA or HSA as a carrier protein to stabilize the cytokine and prevent adsorption to plastic.
• Mix gently by swirling or gentle pipetting. Do not vortex or pipette vigorously, as this can denature the protein.
• Aliquot the reconstituted solution to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage:
  • Store aliquots at -20°C to -80°C for long-term use.
  • For short-term use (up to 1 week), store at 2–8°C.
• Working dilutions for cell culture should be made in cell culture medium or buffer containing a carrier protein (e.g., 0.1% BSA, 5% HSA, or 10% FBS) to further stabilize the cytokine.
• Recommended working concentrations for bioassays (e.g., TF-1 cell proliferation) are typically in the ng/mL range (e.g., ED₅₀ values of 0.04–2 ng/mL depending on the assay and batch).

Summary protocol:

1. Centrifuge vial, open carefully.
2. Add sterile water or PBS (with or without 0.1% BSA) to achieve 0.1–1.0 mg/mL.
3. Gently mix to dissolve; avoid vortexing.
4. Aliquot and store at -20°C to -80°C.
5. Prepare working dilutions in cell culture medium with carrier protein.

Key notes:

• Always use sterile technique.
• Avoid repeated freeze-thaw cycles.
• Carrier proteins (BSA, HSA, FBS) are strongly recommended for stability and activity.

These steps are broadly applicable for recombinant cytokines and are supported by multiple technical datasheets and protocols.