Recombinant Mouse Thrombopoietin (NS0 Expressed)

Recombinant Mouse Thrombopoietin (NS0-expressed) is a well-characterized, biologically active protein widely used in research applications involving hematopoiesis, megakaryocyte differentiation, and platelet production. Here are the key reasons to use NS0-expressed recombinant mouse thrombopoietin in your research:

1. High Biological Activity

• NS0-expressed recombinant mouse TPO is fully biologically active and has been validated in cell proliferation assays (e.g., using MO7e human megakaryocytic leukemic cells), with an ED50 typically in the range of 5–15 ng/mL.
• It effectively stimulates the proliferation, maturation, and differentiation of megakaryocytes, making it ideal for studies of thrombopoiesis and megakaryocyte biology.

2. Serum-Free Compatibility

• The NS0-expressed form is specifically recommended for serum-free megakaryocyte colony-stimulating assays. This is important for experiments where serum components could interfere with results or introduce variability.
• It allows for precise control over culture conditions, which is essential for reproducible and interpretable data in stem cell and hematopoietic differentiation studies.

3. Consistency and Purity

• Recombinant proteins produced in NS0 cells are generally highly purified and consistent between batches, reducing experimental variability.
• This is particularly important for quantitative assays, dose-response studies, and functional characterization.

4. Broad Research Applications

• Megakaryocyte and platelet studies: TPO is the primary regulator of megakaryocyte development and platelet production.
• Hematopoietic stem cell (HSC) research: TPO supports the survival, self-renewal, and expansion of HSCs and primitive multilineage progenitors.
• Ex vivo expansion: Useful for expanding hematopoietic stem and progenitor cells for transplantation or functional studies.
• Differentiation assays: Supports the differentiation of pluripotent stem cells into megakaryocytes and platelets.

5. Well-Established Use in the Literature

• NS0-expressed TPO is widely cited in peer-reviewed studies and is considered a gold standard for in vitro and in vivo hematopoietic research.
• It is compatible with a variety of downstream applications, including bioassays, ELISA standards, and functional cell-based assays.

6. Species-Specific Activity

• Mouse TPO is specifically active on mouse cells, making it the preferred choice for mouse models, primary mouse cell cultures, and studies involving mouse hematopoietic systems.

In summary: Use Recombinant Mouse Thrombopoietin (NS0-expressed) when you require a highly active, serum-free compatible, and consistent source of TPO for studies involving megakaryocyte differentiation, platelet production, hematopoietic stem cell expansion, or functional assays in mouse systems.

Yes, you can use recombinant mouse thrombopoietin (NS0 expressed) as a standard for quantification or calibration in your ELISA assays, provided it is compatible with your assay system.

Supporting details and best practices:

• Recombinant mouse thrombopoietin produced in NS0 cells is commonly used as a standard in commercial mouse thrombopoietin ELISA kits and for assay calibration. These standards are typically validated for use in quantitative ELISA, allowing accurate interpolation of sample concentrations from a standard curve.
• The standard curve in ELISA is generated using serial dilutions of the recombinant protein standard, and sample concentrations are calculated based on this curve. The recombinant standard should be of high purity and well-characterized, with its concentration accurately determined.
• NS0-expressed recombinant proteins are widely accepted for ELISA calibration because their post-translational modifications (such as glycosylation) are similar to those of native mouse proteins, which can be important for antibody recognition in sandwich ELISAs.
• It is important to ensure that the recombinant standard matches the analyte detected by your ELISA antibodies (i.e., same isoform, similar glycosylation, and epitope accessibility). Most commercial ELISA kits are validated to detect both natural and recombinant mouse thrombopoietin, including NS0-expressed forms.
• For best results:
  • Reconstitute and store the recombinant standard as recommended by the manufacturer or protocol.
  • Prepare a fresh standard curve for each assay run to ensure accuracy.
  • Confirm that your ELISA kit or custom assay is validated for use with recombinant mouse thrombopoietin as a standard. Most kits specify this in their documentation.

Limitations and considerations:

• If your ELISA kit is designed for a different expression system (e.g., E. coli-expressed TPO), minor differences in glycosylation or folding may affect antibody binding, though this is uncommon for well-validated kits.
• Always check the specificity and cross-reactivity information in your ELISA kit documentation to ensure compatibility with your recombinant standard.

In summary, NS0-expressed recombinant mouse thrombopoietin is suitable and widely used as a standard for ELISA quantification, provided it is compatible with your assay’s antibodies and protocol.

Recombinant Mouse Thrombopoietin (NS0 Expressed) has been validated in published research for several key applications, primarily in studies involving hematopoietic and megakaryocytic cell biology.

Validated Applications:

• Megakaryocyte Proliferation and Differentiation: Used to stimulate proliferation and maturation of megakaryocytes in vitro, often assessed via cell proliferation assays using cell lines such as MO7e.
• Expansion of Hematopoietic Stem and Progenitor Cells: Frequently combined with other cytokines (e.g., stem cell factor, Flt3 ligand) to promote the survival, self-renewal, and expansion of primitive hematopoietic cells in culture.
• Platelet Production and Activation: Applied in studies of in vitro platelet activation and thrombopoiesis, including models of thrombocytopenia and platelet recovery.
• Apoptosis Assays: Utilized to investigate the effects of thrombopoietin on cell survival and apoptosis, particularly in hematopoietic lineages.
• Functional Bioactivity Assays: Validated by its ability to stimulate cell proliferation in bioactivity assays, especially using MO7e human megakaryocytic leukemic cells, with defined ED50 values for activity.
• Control and Reference Protein: Used as a control or reference standard in various experimental setups to validate cytokine-dependent cellular responses.

Additional Context:

• In vitro studies have demonstrated that recombinant mouse thrombopoietin supports the expansion and maintenance of hematopoietic stem cells and multilineage progenitors, making it a critical reagent for stem cell research and regenerative medicine protocols.
• It is also employed in combination with other growth factors to optimize culture conditions for hematopoietic cell expansion and differentiation.
• The protein’s bioactivity is routinely validated by its capacity to induce proliferation in specific cell lines, ensuring its suitability for mechanistic and therapeutic studies.

Summary Table of Applications

These applications are supported by both product documentation and published research, confirming the utility of NS0-expressed recombinant mouse thrombopoietin in hematopoietic and megakaryocytic cell biology.

To reconstitute and prepare Recombinant Mouse Thrombopoietin (NS0 Expressed) for cell culture experiments, dissolve the lyophilized protein in sterile buffer—typically sterile PBS or distilled water—to a working concentration of 50 μg/mL to 1 mg/mL, then dilute further in cell culture medium containing a carrier protein such as BSA or serum.

Step-by-step protocol:

1. Preparation:

   • Briefly centrifuge the vial to collect all lyophilized protein at the bottom before opening.
   • Warm the vial to room temperature before opening to minimize condensation.

2. Reconstitution:

   • Add sterile PBS or sterile distilled water to achieve a concentration between 50 μg/mL and 1 mg/mL. For NS0-expressed TPO, 50 μg/mL in sterile PBS is commonly recommended.
   • Gently mix by pipetting; do not vortex or shake vigorously to avoid protein denaturation.
   • If solubility issues arise, incubate the solution at 4°C overnight.

3. Aliquoting and Storage:

   • Divide the reconstituted protein into small aliquots to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C or –70°C in a manual defrost freezer.
   • Avoid multiple freeze-thaw cycles, as this can degrade the protein.

4. Dilution for Cell Culture:

   • For cell culture, dilute the stock solution in medium containing a carrier protein (e.g., 0.1–1% BSA or FCS) to minimize adsorption and loss.
   • Use low endotoxin medium for sensitive cell types.
   • Filter-sterilize the final working solution if required, using a 0.22 μm low-protein-binding filter.

5. Working Concentration:

   • The optimal concentration for cell culture should be determined empirically, but typical bioassay concentrations range from 0.1–10 ng/mL depending on cell type and experimental design.

Additional notes:

• Always consult the specific product datasheet for recommended buffer and concentration, as formulations may vary.
• For functional assays, confirm protein activity (e.g., via proliferation assay or SDS-PAGE).
• Carrier-free preparations may require extra care to prevent protein loss due to adsorption; always include carrier protein in working solutions.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant mouse thrombopoietin for cell culture applications.