Recombinant Mouse Eotaxin-2

Recombinant Mouse Eotaxin-2 is used in research applications to study the mechanisms of eosinophil and basophil chemotaxis, type 2 immune responses, and hematopoietic regulation in mouse models and cell-based assays.

Key scientific reasons to use recombinant Mouse Eotaxin-2 include:

• Chemotaxis Studies: Eotaxin-2 is a CC chemokine that specifically binds to the CCR3 receptor, inducing chemotaxis of eosinophils, basophils, Th2 T cells, and certain dendritic cell subsets. This makes it essential for dissecting the cellular migration and recruitment mechanisms in allergic inflammation, asthma, and other Th2-driven diseases.

• Mediator Release Assays: It stimulates the release of histamine and leukotriene C4 from basophils primed with IL-3, allowing investigation of mediator release in allergic responses.

• Hematopoietic Regulation: Eotaxin-2 inhibits the proliferation of multipotential hematopoietic progenitor cells, providing a tool to study hematopoietic suppression and myeloid cell regulation.

• Type 2 Immunity: Its production is upregulated by allergen challenge and IL-4 overexpression, linking it to Th2 cytokine signaling and allergic inflammation.

• ELISA Standardization: Recombinant Mouse Eotaxin-2 (especially aa 27-119) is suitable as a standard for quantification or calibration in ELISA assays, enabling precise measurement of Eotaxin-2 levels in biological samples.

• Reproducibility and Mechanistic Relevance: Using recombinant protein ensures batch-to-batch consistency and mechanistically relevant activity, critical for reproducible in vitro and in vivo experiments.

• Cellular Activation and Differentiation: It can be used to activate or polarize specific immune cell populations in culture, supporting studies on cytokine production, cell expansion, and differentiation.

Typical applications include:

• Chemotaxis assays (e.g., transwell migration)
• Cell activation and mediator release studies
• ELISA calibration and quantification
• Hematopoietic progenitor cell proliferation assays
• Mouse models of allergic inflammation and asthma

Best practices: Use recombinant Mouse Eotaxin-2 at validated concentrations for your specific assay (e.g., 10–100 ng/mL for chemotaxis), confirm compatibility with your detection system (especially for ELISA), and ensure endotoxin levels are sufficiently low for cell-based assays.

In summary, recombinant Mouse Eotaxin-2 is a mechanistically precise reagent for dissecting eosinophil biology, type 2 immunity, and hematopoietic regulation in murine systems, supporting both basic and translational immunology research.

Yes, recombinant Mouse Eotaxin-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly quantified, and compatible with your assay system. This is a common practice in ELISA development and commercial kits.

Key considerations and supporting details:

• Recombinant protein standards are routinely used in ELISA kits for generating standard curves, allowing for the quantification of the target analyte in biological samples. Commercial Mouse Eotaxin-2 (CCL24) ELISA kits typically include a recombinant Mouse Eotaxin-2 standard, often produced in E. coli and spanning the mature protein sequence.
• Standard curve preparation: The recombinant standard is serially diluted to generate a standard curve, which is then used to interpolate the concentrations of Eotaxin-2 in unknown samples.
• Equivalence to native protein: Recombinant standards are validated to ensure they mimic the native protein in immunoreactivity and assay performance, which is critical for accurate quantification.
• Carrier-free vs. carrier-added: For ELISA calibration, a carrier-free recombinant protein is generally preferred to avoid interference, unless your protocol or kit specifies otherwise.
• Validation: Ensure the recombinant Mouse Eotaxin-2 you use is validated for ELISA applications and that its concentration is accurately determined (e.g., by BCA or absorbance at 280 nm).

Best practices:

• Confirm the recombinant protein’s sequence and post-translational modifications match those recognized by your ELISA antibodies.
• Prepare and store the standard according to best practices (aliquot, avoid freeze-thaw cycles, use appropriate diluents).
• Run the standard in parallel with your samples in each assay to account for any lot-to-lot variation.

Summary:
Using recombinant Mouse Eotaxin-2 as a standard is scientifically appropriate and aligns with standard ELISA protocols, as long as the protein is of high quality and compatible with your assay system.

Recombinant Mouse Eotaxin-2 has been validated for several key applications in published research, primarily related to its role as a chemokine in immunological studies.

Validated Applications:

• Functional Assays:
  Used to assess chemotactic activity, especially for eosinophils, basophils, Th2 T cells, and certain dendritic cell subsets. The protein’s ability to induce chemotaxis is a standard validation method, often measured by migration assays using transfected cell lines expressing CCR3.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Recombinant Mouse Eotaxin-2 serves as a standard or analyte in ELISA kits for quantifying eotaxin-2 levels in biological samples such as plasma, cell culture supernatants, and tissue lysates.

• Blocking/Neutralization Assays:
  Used to block or neutralize eotaxin-2 activity in vitro, helping to dissect its specific role in chemokine signaling and cell migration.

• Cell Analysis Methods:
  Applied in cell-based assays to study effects on hematopoietic progenitor cell proliferation and colony formation, as well as to analyze cytokine-induced chemokine production in monocytes and macrophages.

• Western Blot:
  Validated for use as a positive control or standard in Western blotting to detect eotaxin-2 protein expression.

Supporting Details:

• Chemotaxis Assays:
  The protein’s biological activity is routinely validated by its ability to induce migration of CCR3-expressing cells, such as BaF/3 cells transfected with mCCR3, or primary eosinophils.

• Suppression of Colony Formation:
  Eotaxin-2 is a strong suppressor of colony formation by multipotential hematopoietic progenitor cell lines, which is another functional validation.

• Immunological Studies:
  Used to study the regulation of eotaxin-2 production by cytokines (e.g., IL-1β, LPS, IL-4) in monocytes and macrophages, elucidating its role in both innate and adaptive immunity.

Summary Table of Validated Applications

Note:
All applications are for laboratory research use only and not for diagnostic or therapeutic purposes.

If you require protocols or best practices for any specific application, please specify the intended experimental context.

To reconstitute and prepare Recombinant Mouse Eotaxin-2 (CCL24) protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) or sterile ultrapure water to a concentration of at least 100 µg/mL. For optimal stability and biological activity, follow these technical steps:

Reconstitution Protocol:

• Add sterile PBS (pH 7.2–7.4) or sterile 18 MΩ-cm H₂O directly to the lyophilized protein to achieve a final concentration of ≥100 µg/mL.
• Gently mix by pipetting or swirling; avoid vigorous vortexing to prevent protein denaturation.
• If the protein is difficult to dissolve, allow it to sit at room temperature for 10–15 minutes, then gently mix again.
• If recommended by the supplier or for long-term storage, add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to minimize adsorption and aggregation.

Preparation for Cell Culture:

• After reconstitution, further dilute the stock solution in cell culture medium or assay buffer to the desired working concentration (typically 10–100 ng/mL for chemotaxis assays, but refer to your experimental protocol).
• Filter-sterilize the final working solution if necessary, using a 0.2 µm filter, especially if sterility is critical for cell culture.
• Avoid repeated freeze-thaw cycles, as these can reduce protein activity and stability.

Storage:

• Store the reconstituted protein at 2–8°C for short-term use (up to one month).
• For long-term storage, aliquot and freeze at –20°C to –70°C in a manual defrost freezer.
• Always avoid repeated freeze-thaw cycles; aliquot into single-use volumes if possible.

Additional Notes:

• Check the specific product datasheet for any unique instructions, as formulation buffers (e.g., acetonitrile/TFA vs. PBS) may affect solubility and compatibility with cell culture.
• Confirm the endotoxin level is suitable for cell culture (<0.01 EU/µg is typical for recombinant chemokines).
• The biological activity is typically measured by chemotaxis assays using CCR3-expressing cells; recommended working concentrations are 10–100 ng/mL.

Summary Table:

Always refer to your specific batch’s certificate of analysis or datasheet for any unique instructions. If your protein was lyophilized from acetonitrile/TFA, ensure complete removal or dilution before cell culture to avoid cytotoxicity.