Recombinant Human Eotaxin

Recombinant Human Eotaxin (CCL11) is widely used in research applications to study eosinophil biology, allergic inflammation, and chemokine signaling due to its potent and specific chemoattractant activity for eosinophils. It enables controlled, reproducible experiments investigating cellular responses, signaling pathways, and disease mechanisms relevant to immunology and inflammation.

Key scientific reasons to use recombinant human Eotaxin in research include:

• Eosinophil Chemotaxis and Activation: Eotaxin is a highly selective chemoattractant for eosinophils, making it essential for in vitro chemotaxis assays, functional studies of eosinophil migration, and activation. It induces oxygen radical production, calcium mobilization, actin reorganization, and upregulation of activation markers in human eosinophils.

• Modeling Allergic and Inflammatory Responses: Eotaxin is upregulated in tissues during allergic inflammation (e.g., asthma), and its recombinant form allows for mechanistic studies of eosinophil recruitment and activation in disease models. It is used to mimic the inflammatory milieu and dissect the roles of chemokines in airway and tissue responses.

• Cell Signaling and Pathway Analysis: Recombinant Eotaxin enables investigation of downstream signaling events, such as CCR3 receptor activation, ERK pathway involvement, and gene expression changes in target cells (e.g., fibroblasts, epithelial cells, immune cells).

• Bioassays and ELISA Standards: It serves as a standard for quantifying Eotaxin levels in biological samples via ELISA, and as a positive control in bioassays assessing chemokine activity.

• Cellular Senescence and Oxidative Stress: Recent studies show Eotaxin promotes reactive oxygen species (ROS) production and cellular senescence in human fibroblasts, expanding its utility to aging and stress response research.

• Cancer and Tissue Remodeling: Eotaxin has been implicated in tumor cell migration, invasion, and tissue remodeling, allowing researchers to explore its role in cancer biology and fibrosis.

• Reproducibility and Purity: Recombinant proteins provide high purity and batch-to-batch consistency, critical for quantitative and mechanistic studies.

Typical applications include:

• Chemotaxis assays
• Cell signaling studies
• ELISA standardization
• In vitro modeling of allergic inflammation
• Analysis of eosinophil function and activation
• Investigation of tissue remodeling and fibrosis

Using recombinant human Eotaxin ensures experimental control and specificity, facilitating robust mechanistic insights into eosinophil-driven processes and chemokine biology.

Yes, recombinant human Eotaxin can be used as a standard for quantification or calibration in ELISA assays, provided it is of appropriate purity and formulation. Multiple sources confirm that purified recombinant human Eotaxin is commonly used as a quantitative standard in ELISA protocols for measuring human Eotaxin protein levels.

Key considerations and supporting details:

• Scientific precedent: Published protocols and commercial ELISA kits routinely use recombinant human Eotaxin as the standard for generating calibration curves. For example, a peer-reviewed study describes the development of a sandwich ELISA using purified recombinant Eotaxin as the standard for quantification.

• Formulation: Ensure the recombinant Eotaxin is formulated for ELISA use. Some preparations include carrier proteins (e.g., BSA) to stabilize the protein and prevent adsorption to plasticware, which is beneficial for standard preparation. Carrier-free formulations are also available but may require additional precautions to prevent protein loss.

• Reconstitution and dilution: Follow the manufacturer’s or supplier’s instructions for reconstitution and dilution to achieve accurate concentrations for your standard curve. For example, a typical protocol may involve reconstituting lyophilized recombinant Eotaxin in sterile water or buffer to a defined concentration, then preparing serial dilutions for the standard curve.

• Validation: It is important to verify that the recombinant standard produces a dose-response curve parallel to that of the endogenous analyte in your sample matrix. This ensures accurate quantification and accounts for potential matrix effects.

• Application-specific notes: Some recombinant proteins are specifically labeled for use as ELISA standards and not for bioassays, and vice versa. Confirm that your recombinant Eotaxin is intended for use as an ELISA standard.

Summary of best practices:

• Use recombinant human Eotaxin of high purity, validated for ELISA.
• Prepare and dilute standards according to protocol, using appropriate buffers.
• Confirm parallelism between standard and sample curves.
• Store and handle the recombinant protein as recommended to maintain stability.

In conclusion, recombinant human Eotaxin is widely accepted and validated as a standard for ELISA quantification, provided you follow best practices for preparation and validation in your specific assay system.

Recombinant Human Eotaxin (CCL11) has been validated in published research for a range of applications, primarily involving functional cell-based bioassays, ELISA (as a standard), binding assays, and mechanistic studies of eosinophil and immune cell function.

Key validated applications include:

• Bioassays:
  Widely used to assess chemotactic activity, cell signaling, and functional responses in various human cell types, especially eosinophils, basophils, fibroblasts, and airway epithelial cells. Examples include:

  • Chemoattraction and migration assays for eosinophils and neoplastic glial cells.
  • Analysis of cell invasion, degranulation, cytokine synthesis, and survival in immune cells.
  • Investigation of signaling pathways (e.g., CCR3-ERK, ROS production, senescence markers) in fibroblasts and epithelial cells.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Used as a standard for quantifying eotaxin levels in biological samples such as plasma and breast milk.

• Binding Assays:
  Applied to study receptor-ligand interactions, particularly with CCR3 on immune and airway epithelial cells.

• Western Blot:
  Validated for detection and quantification of eotaxin protein in cell lysates and supernatants.

• Blocking Assays:
  Used to assess the inhibition of eotaxin-mediated effects, often to dissect signaling pathways or receptor specificity.

Representative published research applications:

• Induction of cellular senescence and oxidative stress:
  Recombinant human eotaxin has been shown to induce reactive oxygen species (ROS) production and senescence-associated markers in human lung fibroblasts.
• Eosinophil activation:
  Demonstrated to stimulate oxygen radical production, calcium mobilization, actin reorganization, and upregulation of activation markers in human eosinophils.
• Cancer cell invasion:
  Used to study the promotion of prostate cancer cell invasion via CCR3-ERK pathway activation.
• Immunomodulation:
  Investigated for its role in inflammatory responses, including effects on basophil migration and cytokine synthesis, and as a marker in allergic inflammation and asthma.

Summary Table of Validated Applications:

These applications are supported by both product validation data and peer-reviewed publications, demonstrating the protein’s utility in immunology, cell biology, and disease mechanism studies.

To reconstitute and prepare Recombinant Human Eotaxin (CCL11) for cell culture experiments, dissolve the lyophilized protein at a concentration of 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA/HSA) as a carrier protein. This helps stabilize the protein and prevents adsorption to surfaces.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile PBS (or sterile distilled water if specified by the product datasheet) to achieve a final concentration of 100 μg/mL.
• Include 0.1–1% BSA or HSA in the buffer to enhance protein stability, especially for cell culture applications.
• Gently pipette the solution up and down to dissolve. Do not vortex, as vigorous mixing may denature the protein.
• Allow several minutes for complete dissolution at room temperature.
• Aliquot the solution into working volumes to avoid repeated freeze-thaw cycles.
• Storage:
  • Short-term: 2–8 °C for up to one week.
  • Long-term: –20 °C to –70 °C for up to three months.
  • Avoid repeated freeze-thaw cycles to maintain protein integrity.

Additional notes for cell culture:

• Before adding to cells, further dilute the stock solution in cell culture medium containing serum or BSA to minimize adsorption and maintain bioactivity.
• Always use sterile technique to prevent contamination.
• If the protein is used for signaling assays, concentrations of ≥100 ng/mL are commonly effective for functional responses such as ERK phosphorylation.

Summary Table:

Always consult the specific product datasheet for any unique requirements, as formulations may vary between lots or suppliers.