Recombinant Human Eotaxin-3 (CCL-26)

Recombinant Human Eotaxin-3 (CCL-26) is a chemokine widely used in research to study eosinophil and basophil recruitment, allergic inflammation, and chemokine receptor signaling, particularly via CCR3. Its use is justified in applications where precise, reproducible, and species-specific chemotactic or signaling responses are required.

Key reasons to use recombinant human Eotaxin-3 (CCL-26) in research:

• Chemotaxis Assays: Eotaxin-3 is a potent chemoattractant for eosinophils and basophils, making it essential for in vitro and in vivo studies of cell migration, especially in the context of allergic diseases such as asthma, atopic dermatitis, and eosinophilic esophagitis. It is also chemotactic for PHA-activated T cells.

• Receptor Specificity and Signaling: Eotaxin-3 is a selective agonist for CCR3 and a natural antagonist for CCR1, CCR2, and CCR5, allowing for detailed dissection of chemokine receptor signaling pathways and cross-desensitization studies. This is particularly relevant for understanding the molecular mechanisms of inflammation and immune cell trafficking.

• Disease Modeling: Elevated CCL-26 levels are associated with eosinophilic infiltration in tissues and are implicated in diseases such as asthma, chronic rhinosinusitis, and eosinophilic esophagitis. Recombinant Eotaxin-3 enables modeling of these pathologies and the testing of therapeutic interventions targeting eosinophil recruitment.

• Biomarker Studies: Plasma and tissue levels of Eotaxin-3 correlate with mucosal eosinophil infiltration and disease severity, making it a valuable tool for biomarker validation and mechanistic studies in translational research.

• Structure-Function Analysis: Recombinant Eotaxin-3 allows for mutagenesis and structure-function studies, such as investigating the importance of the N-terminal domain for CCR3 activation and chemotactic activity.

• Standardization and Reproducibility: Using recombinant protein ensures batch-to-batch consistency, defined activity, and the absence of confounding contaminants, which is critical for reproducible experimental results.

• Immunoregulatory Studies: Eotaxin-3 is involved in broader immunoregulatory and inflammatory processes, including modulation of T cell chemotaxis and antagonism of other chemokine receptors, supporting its use in studies of immune cell interactions and cytokine networks.

In summary, recombinant human Eotaxin-3 (CCL-26) is a versatile reagent for dissecting the mechanisms of eosinophil and basophil recruitment, chemokine receptor biology, and the pathogenesis of allergic and inflammatory diseases, as well as for developing and validating new therapeutic strategies.

Yes, recombinant human Eotaxin-3 (CCL-26) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known.

Supporting details:

• Commercial ELISA kits for human Eotaxin-3 routinely use recombinant Eotaxin-3, typically expressed in E. coli, as the calibration standard. The calibration of these assays is explicitly stated to be against highly purified recombinant human Eotaxin-3.
• The standard curves generated with recombinant Eotaxin-3 are parallel to those obtained with natural Eotaxin-3, indicating that the recombinant protein is suitable for quantification purposes in ELISA.
• Recombinant Eotaxin-3 is validated for use in ELISA applications, as indicated by multiple sources.
• When preparing your standard, ensure you follow the recommended reconstitution protocol (e.g., reconstituting in PBS with carrier protein such as BSA if required) and use the same diluent as your samples to minimize matrix effects.

Best practices:

• Use a recombinant Eotaxin-3 standard that matches the isoform and sequence recognized by your ELISA antibodies.
• Confirm that the recombinant protein is carrier-free or contains only inert carriers (such as BSA) that do not interfere with the assay.
• Prepare the standard curve in the same matrix as your samples (e.g., serum, plasma, or culture medium) for optimal accuracy.
• Store and handle the recombinant standard according to the manufacturer’s instructions to maintain stability and activity.

Limitations:

• If your ELISA is designed for diagnostic use, ensure the recombinant standard is validated for that specific application, as most are for research use only.
• Always check for potential differences in glycosylation or folding between recombinant and natural proteins, though for Eotaxin-3, E. coli-expressed protein is widely accepted for calibration.

In summary, recombinant human Eotaxin-3 is appropriate and commonly used as a standard for ELISA quantification, provided it is compatible with your assay system and prepared according to validated protocols.

Recombinant Human Eotaxin-3 (CCL-26) has been validated in published research for several key applications, primarily related to its biological activity and role in chemokine signaling. The main applications supported by peer-reviewed studies include:

1. Chemotaxis Assays:
   Eotaxin-3/CCL-26 has been shown to induce migration (chemotaxis) of eosinophils, basophils, and Th2-type T lymphocytes. It is particularly effective at recruiting eosinophils from asthmatic patients, with studies demonstrating its potency compared to other eotaxins (CCL11 and CCL24) in human eosinophil migration assays.

   • Reference: CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2 (PubMed ID: 23532518).

2. Calcium Flux Assays:
   Eotaxin-3/CCL-26 induces calcium flux in eosinophils and CCR3-transfected cells, indicating its ability to activate intracellular signaling pathways downstream of CCR3.

   • Reference: R&D Systems product background and related citations.

3. Binding Assays:
   Eotaxin-3/CCL-26 binds to CCR3, as well as to CCR1, CCR2, and CCR5, acting as both an agonist and antagonist depending on the receptor. Binding assays have been used to characterize its interaction with these receptors.

   • Reference: Eotaxin-3/CCL26 is a natural antagonist for CC chemokine receptors CCR1, CCR2, and CCR5 (PubMed ID: 15039444).

4. Functional Modulation of Cellular Recruitment:
   Studies have shown that Eotaxin-3/CCL-26 can modulate cellular recruitment by attracting Th2 lymphocytes, eosinophils, and basophils via CCR3, while simultaneously blocking recruitment of Th1 lymphocytes and monocytes via CCR1, CCR2, and CCR5.

   • Reference: Eotaxin-3/CCL26 is a natural antagonist for CC chemokine receptors CCR1, CCR2, and CCR5 (PubMed ID: 15039444).

5. Bioassays and ELISA Standards:
   Recombinant Eotaxin-3/CCL-26 is used as a standard in ELISA assays and in various bioassays to measure chemokine activity and receptor engagement.

   • Reference: R&D Systems product documentation and Assay Genie product details.

6. Gene Expression and Protein Expression Studies:
   Eotaxin-3/CCL-26 expression has been studied in response to cytokines such as IL-4 and IL-13, particularly in vascular endothelial cells and lung epithelial cells.

   • Reference: Activation of eotaxin-3/CCL26 gene expression in human dermal fibroblasts (PubMed ID: 11544308).

In summary, Recombinant Human Eotaxin-3 (CCL-26) has been validated for use in chemotaxis, calcium flux, receptor binding, functional modulation of immune cell recruitment, bioassays, ELISA standards, and gene/protein expression studies.

To reconstitute and prepare Recombinant Human Eotaxin-3 (CCL-26) protein for cell culture experiments, follow these general best practices based on manufacturer guidelines and scientific protocols:

1. Reconstitution

• Centrifuge the lyophilized protein vial briefly before opening to ensure all powder is at the bottom.
• Reconstitute the protein in sterile PBS (phosphate-buffered saline) or sterile distilled water, typically to a concentration of 0.1 mg/mL (100 µg/mL). Some suppliers recommend using PBS with 0.1% BSA (bovine serum albumin) to stabilize the protein and prevent adsorption to surfaces.
• Gently pipette the solution up and down or swirl the vial to dissolve the protein. Do not vortex, as this may denature the protein.
• Allow several minutes for complete reconstitution at room temperature.

2. Storage

• After reconstitution, the protein can be stored at 2–8°C for short-term use (up to 1 week).
• For long-term storage, aliquot the protein and store at –20°C to –80°C in a manual defrost freezer. Avoid repeated freeze-thaw cycles, as this can reduce activity and stability.
• If storing at low temperatures, consider adding 0.1% BSA or another carrier protein to prevent loss due to adsorption.

3. Preparation for Cell Culture

• Prior to use in cell culture, dilute the reconstituted protein to the desired working concentration in cell culture medium or buffer.
• Filter-sterilize the solution (0.22 µm filter) if required for cell culture applications.
• Ensure the final concentration of BSA (if used) is compatible with your cell type and assay.

4. Handling Tips

• Always use sterile technique to prevent contamination.
• Use low-binding tubes and pipette tips to minimize protein loss.
• Refer to the lot-specific datasheet for any product-specific instructions or recommendations.

Example Protocol (based on common supplier recommendations):

1. Centrifuge vial for 1 minute at 1000 x g.
2. Add 100 µL sterile PBS (with 0.1% BSA if desired) to a 10 µg vial to achieve 100 µg/mL.
3. Gently mix until fully dissolved.
4. Aliquot and store at –80°C for long-term use.
5. Thaw aliquots on ice and use immediately for cell culture experiments.

Always consult the specific product datasheet for any unique requirements or recommendations.