Recombinant Mouse RELMα

Recombinant Mouse RELMα is widely used in research to investigate immune regulation, tissue remodeling, and disease mechanisms, particularly in murine models of inflammation, fibrosis, infection, and pulmonary hypertension.

Key scientific applications and rationale include:

• Modeling Immune Responses: RELMα is a marker and effector of alternatively activated (M2) macrophages, which play critical roles in modulating inflammation, limiting Th2 responses, and promoting Th17 or regulatory T cell responses. Recombinant RELMα allows for controlled in vitro and in vivo studies of macrophage polarization and function.

• Studying Tissue Remodeling and Fibrosis: RELMα contributes to extracellular matrix remodeling, myofibroblast differentiation, and smooth muscle cell proliferation, especially in lung tissue. It is implicated in the pathogenesis of pulmonary fibrosis and pulmonary hypertension, making recombinant RELMα valuable for dissecting these processes and testing therapeutic interventions.

• Investigating Host Defense and Infection: RELMα-expressing macrophages are essential for protection against fatal lung damage and for controlling parasite burden during helminth infection. Recombinant RELMα can be used to study immune cell recruitment, activation, and the resolution of inflammation in infection models.

• Mechanistic Studies: Recombinant RELMα enables precise mechanistic studies, such as:

  • Activation of signaling pathways (e.g., PI3K/AKT, Notch1/Jagged1, BTK).
  • Modulation of damage-associated molecular pattern (DAMP) signaling, including HMGB1/RAGE axis in macrophages.
  • Chemotaxis assays and immune cell functional assays.

• Protocol Standardization: Using recombinant protein ensures reproducibility and specificity in experimental setups, such as cell culture stimulation, ELISA standards, or in vivo administration.

Best practices for using recombinant RELMα:

• Validate biological activity in your specific assay system.
• Use appropriate controls, such as vehicle or unrelated recombinant proteins.
• Optimize concentration and exposure time for your cell type or animal model.

In summary, recombinant mouse RELMα is a versatile tool for dissecting the molecular and cellular mechanisms underlying immune regulation, tissue remodeling, and disease progression in mouse models, with broad relevance to immunology, pathology, and translational research.

Yes, recombinant Mouse RELMα can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for this purpose.

Key considerations and supporting details:

• Recombinant proteins are commonly used as ELISA standards when purified native protein is unavailable or impractical to obtain. The recombinant Mouse RELMα, if of high purity and accurately quantified, is suitable for generating a standard curve in ELISA assays targeting this analyte.

• ELISA kits for Mouse RELMα often use recombinant protein as the standard, and these kits are validated to ensure that both natural and recombinant forms are recognized equivalently by the assay antibodies. This supports the use of recombinant RELMα as a calibrator.

• Validation is critical:

  • You must confirm that the recombinant standard behaves similarly to the endogenous protein in your sample matrix. This is typically assessed by parallelism (ensuring that serial dilutions of samples and standards yield parallel curves) and dilution linearity (recovery of spiked recombinant protein in the sample matrix).
  • The specificity of the antibodies used in your ELISA must be confirmed to recognize both natural and recombinant RELMα without cross-reactivity to related proteins.

• Standard preparation:

  • The recombinant protein should be reconstituted and diluted according to best practices for ELISA standards, ensuring accurate concentration and stability.
  • If possible, use a recombinant standard that has been calibrated against an international reference standard (e.g., NIBSC or WHO), though such references may not always be available for all proteins.

• Documentation and controls:

  • Include appropriate controls (e.g., spiked samples, in-house controls) to monitor assay performance and ensure the standard curve is reliable for quantification.

In summary:
Recombinant Mouse RELMα is suitable as a standard for ELISA quantification if it is pure, accurately quantified, and validated for parallelism and specificity in your assay system. Always validate the standard in your specific assay context to ensure accurate and reproducible results.

Recombinant Mouse RELMα has been validated for several key applications in published research, primarily in studies of immune regulation, inflammation, tissue repair, and pulmonary disease models.

Validated Applications:

• In vitro chemotaxis assays: Recombinant RELMα has been used to demonstrate chemotactic activity on primary mouse bone marrow myeloid cells, specifically via the Bruton’s tyrosine kinase (BTK) pathway.
• Macrophage activation and polarization studies: RELMα is employed to modulate macrophage responses, including licensing macrophages for DAMP (damage-associated molecular pattern) activation, switching macrophage function from pro-inflammatory to proliferative, and influencing M1/M2 polarization.
• Pulmonary hypertension and vascular remodeling models: RELMα is used in mouse models to induce pulmonary hypertension and study its role in vascular remodeling and immune-vascular interactions.
• Tissue repair and fibrosis assays: Recombinant RELMα is applied in ex vivo and in vivo models to assess its impact on epithelial repair, extracellular matrix remodeling, and collagen cross-linking, especially following helminth infection or lung injury.
• Antibacterial activity assays: RELMα has been validated for disrupting bacterial membranes and demonstrating direct antibacterial properties in vitro.
• Immunopathology and cytokine response studies: RELMα is used to investigate its role in limiting type 2 cytokine immunopathology, controlling CD4^+ T cell polarization, and promoting anti-inflammatory responses.
• Reporter and knockout mouse validation: Recombinant RELMα is used to validate gene knockout and reporter mouse models, confirming its expression and functional replacement in transgenic systems.

Additional Context:

• RELMα is frequently used in studies involving macrophage regulatory mechanisms, lung inflammation, and helminth infection models to dissect its role in immune cell recruitment, tissue protection, and resolution of inflammation.
• In pulmonary research, RELMα is a critical mediator of extracellular matrix gene induction and smooth muscle cell proliferation, relevant for modeling chronic lung diseases.
• Its use in ex vivo tissue repair models and flow cytometry validation highlights its utility in both functional and mechanistic studies of immune and epithelial cell biology.

These applications are supported by multiple peer-reviewed studies, confirming the utility of recombinant mouse RELMα in diverse experimental settings related to immunology, tissue biology, and disease modeling.

To reconstitute and prepare Recombinant Mouse RELMα protein for cell culture experiments, centrifuge the vial briefly, then dissolve the lyophilized protein in sterile distilled water to a final concentration of 0.1 mg/mL unless otherwise specified by the product documentation. Avoid vigorous mixing; gently pipette or swirl to ensure complete dissolution.

Detailed protocol:

• Centrifugation: Briefly centrifuge the vial before opening to collect all powder at the bottom.
• Reconstitution: Add sterile distilled water to achieve a concentration of 0.1 mg/mL (e.g., add 100 µL water to 100 µg protein). Gently pipette or swirl; do not vortex.
• Dilution for cell culture: After reconstitution, further dilute the protein in your cell culture medium to the desired working concentration (commonly in the range of 10–200 ng/mL for cell stimulation, but optimize based on your experimental design).
• Carrier protein (optional): For long-term storage or to prevent adsorption, dilute with a solution containing a carrier protein such as 0.1% BSA or 10% FBS. If using serum-free conditions, avoid animal-derived carriers and consider trehalose.
• Aliquoting and storage: Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles. Store aliquots at −20°C or −80°C for long-term use.
• Handling: Always use sterile technique. Equilibrate protein and buffer to room temperature before use. If insoluble particles remain, allow gentle mixing for up to 2 hours at room temperature.

Notes:

• Do not vortex, as this may denature the protein.
• Always consult the Certificate of Analysis or product datasheet for specific instructions, as buffer requirements and optimal concentrations may vary by batch or manufacturer.
• For cell culture, ensure the final solution is endotoxin-free and compatible with your assay conditions.

Example working concentrations: Published studies have used recombinant RELMα at concentrations ranging from 100 nM (approximately 2 µg/mL) for fibroblast stimulation to 200 ng/mL for macrophage activation. Adjust according to your cell type and experimental needs.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant RELMα for cell culture experiments.