Recombinant Mouse IL-3

Recombinant Mouse IL-3 is widely used in research applications because it is a potent cytokine that regulates the survival, proliferation, and differentiation of hematopoietic stem cells and progenitor cells, especially those of the myeloid lineage.

Key scientific applications and rationale include:

• Hematopoietic Cell Culture: Mouse IL-3 is essential for supporting the growth and expansion of hematopoietic stem cells and lineage-committed progenitors in vitro, making it a standard supplement in protocols for bone marrow, spleen, or fetal liver cell cultures.
• Differentiation Studies: It induces differentiation of hematopoietic stem cells into various myeloid cell types, including erythrocytes, megakaryocytes, granulocytes, and macrophages, enabling studies on lineage commitment and cell fate decisions.
• Functional Assays: IL-3 is used in bioassays to stimulate cell proliferation (e.g., NFS-60 cell line), assess cytokine responsiveness, and investigate signaling pathways involved in immune cell activation and growth.
• Immunology and Inflammation Research: IL-3 modulates immune cell functions such as migration, adhesion, phagocytosis, and mediator secretion, making it valuable for studying inflammatory processes, allergic responses, and immune regulation.
• Basophil and Mast Cell Biology: It is the most potent growth factor for basophils and also promotes mast cell growth and differentiation, supporting research into allergy and anaphylaxis mechanisms.
• Receptor Biology: Mouse IL-3 acts through high-affinity IL-3 receptors, transmitting growth and survival signals, which can be exploited to dissect receptor signaling and cytokine-receptor interactions.
• Disease Models: IL-3 is implicated in the pathogenesis of diseases such as leukemia, ulcerative colitis, and asthma, and recombinant protein is used to model these conditions in vitro and in vivo.

Best practices:

• Use recombinant, carrier-free IL-3 for reproducible results and to avoid interference from serum or carrier proteins.
• Optimize concentration for specific cell types and experimental conditions, as biological activity is dose-dependent.

In summary, Recombinant Mouse IL-3 is a versatile tool for hematopoietic, immunological, and disease modeling studies due to its broad effects on cell growth, differentiation, and immune function.

Yes, recombinant mouse IL-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in research ELISAs for cytokines like IL-3.

Supporting details and best practices:

• Recombinant mouse IL-3 is routinely used as a standard in commercial ELISA kits. For example, the Quantikine Mouse IL-3 ELISA uses E. coli-expressed recombinant mouse IL-3 as its standard, and the assay has been validated to quantitate both recombinant and natural mouse IL-3, with parallel dose-response curves for both forms. This demonstrates that recombinant IL-3 is suitable for generating standard curves in ELISA quantification.

• Carrier protein considerations: Recombinant IL-3 is often supplied either with or without carrier proteins (such as BSA). For ELISA standards, it is generally recommended to use the formulation with BSA to improve stability and minimize adsorption to plasticware, unless the presence of BSA would interfere with your assay.

• Concentration accuracy: Ensure the recombinant IL-3 standard is accurately quantified (e.g., by absorbance at 280 nm or amino acid analysis) and properly reconstituted according to the supplier’s instructions. Serial dilutions should be prepared in the same buffer or matrix as your samples to minimize matrix effects.

• Validation: If you are using a recombinant IL-3 standard from a different source than the one used to develop your ELISA kit, it is good practice to verify that the standard curve generated is parallel to the kit’s standard curve, ensuring comparable quantification of natural and recombinant IL-3.

• Research use only: Note that these standards and assays are for research use only and not for diagnostic purposes.

Summary of best practices:

• Use high-purity, well-characterized recombinant mouse IL-3.
• Prepare standards in a matrix similar to your samples.
• Confirm parallelism between recombinant and natural IL-3 curves if possible.
• Store and handle the protein according to manufacturer recommendations to maintain stability.

In conclusion, recombinant mouse IL-3 is widely accepted and validated as a standard for ELISA quantification, provided these technical considerations are followed.

Recombinant Mouse IL-3 has been validated for several key applications in published research, primarily in bioassays, hematopoietic cell culture, and functional studies involving immune cell development and proliferation.

Validated Applications in Published Research:

• Bioassays:
  Recombinant Mouse IL-3 is widely used to stimulate cell proliferation, particularly of the NFS-60 mouse myelogenous leukemia lymphoblast cell line, and to assess cytokine-dependent cell growth. Numerous studies have used it to evaluate the biological activity of compounds or genetic modifications affecting hematopoietic or immune cell function.

• Hematopoietic Stem and Progenitor Cell Research:
  IL-3 is essential for the expansion and differentiation of hematopoietic stem and progenitor cells in vitro. It supports the growth and colony formation of multiple hematopoietic lineages, including myeloid, erythroid, and megakaryocytic cells.

• Macrophage and Basophil Development:
  IL-3 stimulates the clonal growth of pulmonary alveolar macrophages and is a potent growth factor for basophils, supporting their development and function in vitro and in vivo.

• Cell Culture and Differentiation Studies:
  Recombinant Mouse IL-3 is routinely used in cell culture protocols to maintain and differentiate various immune cell types, including mast cells, basophils, and macrophages.

• Functional Assays:
  It is applied in functional studies to investigate cytokine signaling, immune cell activation, and responses to infection or inflammation. For example, it has been used to study the role of IL-3 in leukemia models, immune cell extravasation, and inflammatory responses.

• Immunoassay Standard:
  IL-3 serves as a standard in immunoassays (e.g., ELISA) for quantifying cytokine levels in biological samples.

• Cell Migration, Adhesion, and Motility Assays:
  Published reviews and protocols report its use in assays measuring cell migration, adhesion, and motility, particularly in the context of hematopoietic and immune cell biology.

Representative Published Research Applications:

• Leukemia and Cancer Models:
  Used to support the growth of leukemic progenitor cells and to study drug responses in acute myeloid leukemia and chronic myeloid leukemia models.

• Immune System Mouse Models:
  Applied in genetically engineered mouse models to study human immune cell reconstitution, alveolar macrophage development, and mucosal immune responses.

• Basophil and Mast Cell Biology:
  Validated for studies on basophil extravasation and mast cell development, including functional assays of cytokine-induced gene expression.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed publications, confirming the utility of recombinant Mouse IL-3 in diverse immunological and hematopoietic research contexts.

To reconstitute and prepare Recombinant Mouse IL-3 protein for cell culture experiments, briefly centrifuge the vial to collect the lyophilized powder, then reconstitute in sterile water or PBS to a concentration between 0.1–1.0 mg/mL. Avoid vigorous mixing; gently swirl or invert to dissolve the protein.

Step-by-step protocol:

• Centrifuge the vial for 20–30 seconds to ensure all powder is at the bottom before opening.
• Warm the vial to room temperature before opening to minimize condensation.
• Add sterile water or PBS (consult product datasheet for buffer preference; both are commonly used) to achieve a final concentration of 0.1–1.0 mg/mL.
  • For example, add 100 μL to 1 mg for 1 mg/mL, or 1 mL for 0.1 mg/mL.
• Gently mix by swirling or inverting; do not vortex.
• Allow several minutes for complete dissolution; if solubility issues arise, incubate at 4°C overnight.
• Aliquot the reconstituted solution to minimize freeze-thaw cycles.
• Store aliquots at ≤–20°C for long-term use (up to 6 months), or at 4°C for short-term use (up to 1 week).
• For working dilutions, dilute the stock in cell culture medium or buffer containing a carrier protein such as 0.1–1% BSA or FBS to reduce adsorption and stabilize the protein.

Additional notes:

• Do not exceed 1 mg/mL during reconstitution to avoid solubility issues.
• Avoid repeated freeze-thaw cycles; use aliquots.
• For bioassays, typical working concentrations range from 20–100 pg/mL for cell stimulation, but optimize according to your cell type and experimental design.
• Confirm protein presence and integrity by SDS-PAGE if needed.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant mouse IL-3 for cell culture applications.