Recombinant Mouse Erythropoietin

Recombinant Mouse Erythropoietin (rMuEPO) is a valuable tool for research applications due to its well-characterized biological activity and broad utility in studying hematopoiesis, tissue protection, and immune modulation. Here are several key reasons to use recombinant mouse erythropoietin in your research:

1. Regulation of Erythropoiesis

• rMuEPO is essential for the survival, proliferation, and differentiation of erythroid progenitor cells in mice.
• It prevents apoptosis of erythroid precursors, thereby increasing the production of mature red blood cells (RBCs) and hemoglobin concentration.
• This makes it ideal for studies involving anemia, hematopoietic stem cell differentiation, and bone marrow biology.

2. Tissue-Protective Effects

• Beyond its hematopoietic role, rMuEPO exhibits tissue-protective properties, including anti-apoptotic, antioxidant, and anti-inflammatory effects.
• It has been shown to protect tissues in models of ischemia, hypoxia, and injury, making it useful for research in neuroprotection, cardiovascular disease, and organ injury.

3. Neuroprotective and Neuroregenerative Applications

• rMuEPO can stimulate neurogenesis, reduce neuronal apoptosis, and improve cognitive outcomes in animal models of brain injury.
• It is widely used in neuroscience research to study mechanisms of neuroprotection and repair.

4. Immune Modulation

• Erythropoietin has immunomodulatory effects, including enhancement of immune responses such as increased immunoglobulin production and splenocyte proliferation in mice.
• This property is relevant for studies on immune system regulation and inflammatory diseases.

5. Bone and Muscle Research

• rMuEPO has been shown to prevent bone loss in osteonecrosis and improve skeletal muscle microcirculation and tissue oxygenation.
• It is useful for investigating musculoskeletal disorders and tissue regeneration.

6. Species-Specific Studies

• Using recombinant mouse erythropoietin ensures compatibility with mouse models, providing physiologically relevant results in preclinical studies.
• It is particularly important for in vivo experiments where species-specific receptor interactions are critical.

7. Standardization and Reproducibility

• Recombinant forms are highly purified and standardized, ensuring consistent activity across experiments.
• This is crucial for reproducibility in both in vitro and in vivo research settings.

8. Versatility in Experimental Models

• rMuEPO can be used in a wide range of applications, including cell culture, animal models of disease, and mechanistic studies of signaling pathways.

In summary, recombinant mouse erythropoietin is a versatile and biologically active molecule that supports research in hematopoiesis, tissue protection, neurobiology, immunology, and musculoskeletal biology, making it an essential reagent for studies involving mouse models.

Yes, recombinant Mouse Erythropoietin (EPO) can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system.

Supporting details:

• Many commercial mouse EPO ELISA kits use recombinant mouse EPO as the standard for generating calibration curves, enabling quantification of both recombinant and endogenous EPO in samples.
• ELISA kits are typically validated to ensure that the standard curve generated with recombinant EPO is parallel to the curves obtained with natural EPO from biological samples, confirming that quantification is accurate for both forms.
• The recombinant EPO standard should be of high purity, with a defined concentration, and ideally produced in a system that yields glycosylation patterns similar to native mouse EPO, as glycosylation can affect antibody recognition and assay performance.
• When using recombinant EPO as a standard, ensure that:
  • The ELISA antibodies recognize both recombinant and natural EPO equivalently.
  • The standard is prepared and diluted according to the assay protocol, using the recommended diluent and conditions.
  • The standard curve covers the expected concentration range of your samples.

Best practices:

• Always verify that your specific ELISA kit or custom assay is validated for use with recombinant EPO as a standard. Most sandwich ELISAs for mouse EPO are designed for this purpose.
• Run standards and samples in duplicate or triplicate to ensure accuracy and reproducibility.
• If using a recombinant EPO standard from a source different than the kit manufacturer, confirm its compatibility and equivalence by comparing standard curves and recovery rates.

Limitations:

• Recombinant EPO produced in different expression systems may have varying glycosylation, which can affect antibody binding and quantification. Always use a recombinant standard that closely matches the native protein in your samples.
• For absolute quantification, standards should be calibrated against an international reference, if available.

In summary, recombinant mouse EPO is widely accepted and validated as a standard for ELISA quantification, but assay-specific validation and careful preparation are essential for accurate results.

Recombinant Mouse Erythropoietin (EPO) has been validated in published research primarily for bioassays assessing erythropoietic activity, immunoregulatory studies, and as a standard in ELISA development.

Key validated applications include:

• Bioassays:
  Recombinant mouse EPO is widely used in cell-based bioassays to study erythropoiesis, hematopoietic progenitor cell differentiation, and signaling through the EPO receptor (EPO-R). These assays have been applied to:

  • Investigate cytokine and growth factor receptor signaling in various cell types, including vascular smooth muscle cells, brain, retina, heart, skeletal muscle, kidney, endothelial, and tumor cells.
  • Study JAK2-mediated signaling pathways and receptor dimerization.
  • Assess the effects of EPO on hematopoietic progenitor cell fate, particularly toward erythroid lineage commitment.
  • Model disease states such as osteonecrosis, pyruvate kinase deficiency, and hereditary pulmonary alveolar proteinosis in mice.

• Immunoregulatory and Disease Models:
  Recombinant mouse EPO has been validated in murine models for its immunomodulatory effects, including:

  • Inhibition of T and B cell activation and autoantibody production in lupus models.
  • Prevention of clinical manifestations in autoimmune and inflammatory disease models, such as experimental autoimmune encephalomyelitis, lupus nephritis, and interstitial nephritis.
  • Promotion of regulatory T cell and macrophage differentiation, and enhancement of transplant survival.

• ELISA Development:
  Recombinant mouse EPO is used as a standard for the development and validation of ELISA assays to quantify EPO levels in plasma and other biological samples.

• In Vivo Biological Activity Assays:
  Protocols based on European Pharmacopoeia guidelines use recombinant EPO in mice to assess in vivo erythropoietic activity, often for potency and quality control testing of EPO preparations.

• Other Research Applications:

  • Used in studies of stem cell differentiation, particularly for directing embryonic stem cells toward hematopoietic or erythroid fates.
  • Applied in research on metabolic regulation, such as insulin-stimulated GLUT4 translocation in muscle cells.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and are considered standard in the field for functional and mechanistic research involving erythropoietin in mouse models.

To reconstitute and prepare Recombinant Mouse Erythropoietin (EPO) protein for cell culture experiments, follow these best practices based on manufacturer guidelines and scientific protocols:

1. Reconstitution

• Centrifuge the vial briefly (20–30 seconds in a microcentrifuge) before opening to ensure all lyophilized powder is at the bottom.
• Reconstitute the protein with sterile, distilled water or a recommended buffer (such as sterile PBS). Typical concentrations range from 0.1–1 mg/mL (100–1000 µg/mL), depending on the product and intended use.
  • For example, some protocols recommend reconstituting at 100 µg/mL in sterile PBS with at least 0.1% carrier protein (e.g., human or bovine serum albumin) to stabilize the protein.
  • If using PBS, ensure it is sterile and free of contaminants.
• Gently mix the solution by swirling or inverting the vial. Avoid vigorous shaking or vortexing to prevent foaming and protein denaturation.
• Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

2. Aliquoting and Storage

• Aliquot the reconstituted protein into small, single-use volumes to minimize freeze-thaw cycles.
• Store aliquots at –20°C to –70°C for long-term stability. Avoid repeated freezing and thawing, as this can reduce protein activity.
• For short-term use (within a month), some products can be stored at 2–8°C under sterile conditions.

3. Preparation for Cell Culture

• Thaw aliquots on ice or at 4°C just before use.
• Dilute the protein in appropriate cell culture medium or buffer to the desired working concentration.
• Filter sterilize (0.22 µm filter) if necessary before adding to cell cultures.
• Use immediately after dilution for optimal activity.

4. Additional Tips

• Always refer to the product-specific datasheet or Certificate of Analysis for exact reconstitution instructions, as some recombinant proteins may require specific buffers or additives.
• If the protein is carrier-free, consider adding a carrier protein (e.g., BSA) to prevent adsorption to surfaces and maintain stability.

By following these steps, you can ensure proper reconstitution and preparation of Recombinant Mouse Erythropoietin for reliable results in cell culture experiments.