Recombinant Mouse Flt-3 Ligand

Recombinant Mouse Flt-3 Ligand (FLT3L) is widely used in research to promote the expansion, differentiation, and functional study of hematopoietic and immune cell populations, particularly dendritic cells, early progenitors, and lymphoid lineages.

Key scientific applications and rationale include:

• Expansion and Differentiation of Hematopoietic Progenitors: FLT3L directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells, especially when combined with other cytokines such as IL-3, IL-7, and stem cell factor (SCF). This is essential for studies on hematopoiesis, stem cell biology, and immune reconstitution.

• Generation and Maturation of Dendritic Cells: FLT3L is a potent inducer of dendritic cell (DC) development both in vitro (from bone marrow cultures) and in vivo, making it a critical reagent for immunology research, vaccine development, and studies of antigen presentation. FLT3L-expanded DCs enhance antigen-specific B and T cell responses and can be used to study immune modulation and tolerance.

• Synergistic Effects with Other Cytokines: FLT3L acts synergistically with cytokines such as IL-3, GM-CSF, SCF, IL-2, IL-6, IL-7, and IL-15 to promote the mobilization, proliferation, and differentiation of various hematopoietic and immune cell subsets, including NK cells and B cells.

• Functional Studies in Disease Models: Administration of recombinant FLT3L in mouse models has been shown to:

  • Enhance resistance to infections (e.g., Leishmania major) by expanding DCs and increasing IL-12 production, leading to improved Th1-type immune responses and long-term immunity.
  • Reduce severity of allergic inflammation and support cell-mediated inflammatory responses.
  • Facilitate studies on autoimmune diseases, such as arthritis, by modulating regulatory T cell (Treg) formation and dendritic cell homeostasis.

• Cell Culture and Differentiation Protocols: FLT3L is routinely used in protocols for the in vitro generation of dendritic cells, eosinophils, and other myeloid lineages from mouse bone marrow progenitors, supporting studies in immunology, hematology, and cell therapy development.

• Preclinical and Translational Research: FLT3L is used to mobilize hematopoietic stem and progenitor cells in vivo, which is relevant for transplantation, gene therapy, and regenerative medicine studies.

In summary, using recombinant mouse FLT3L enables precise control over hematopoietic and immune cell development in experimental systems, making it indispensable for basic and translational research in immunology, hematopoiesis, infection, and disease modeling.

Yes, you can use Recombinant Mouse Flt-3 Ligand (Flt3L) as a standard for quantification or calibration in your ELISA assays, provided that your ELISA is designed to detect mouse Flt3L and the recombinant protein is compatible with the assay format.

Key Points Supporting Its Use as an ELISA Standard:

• Functional Activity: Recombinant Mouse Flt-3 Ligand (e.g., R&D Systems 427-FL or similar products) is commonly used as a standard in ELISA kits and bioassays. For example, it is referenced as a standard in several ELISA kits and publications for quantifying mouse Flt3L in biological samples.

• Quantitative Data: The recombinant protein has well-characterized activity and purity. For instance, R&D Systems reports that their recombinant Mouse Flt-3 Ligand stimulates cell proliferation in bioassays with a defined ED50 (0.4–2.4 ng/mL), and it is used to generate standard curves in ELISA protocols.

• Compatibility: Many ELISA kits (such as those from R&D Systems, Proteintech, Abcam, and others) are validated for use with recombinant mouse Flt3L as a standard. The standard curve generated with the recombinant protein allows for accurate quantification of endogenous Flt3L in samples like serum, plasma, or cell culture supernatants.

• Formulation: Recombinant Mouse Flt-3 Ligand is typically supplied lyophilized and can be reconstituted to a known concentration, making it suitable for preparing a dilution series for ELISA standard curves.

Recommendations:

• Check Kit Compatibility: Ensure that the ELISA kit you are using is validated for mouse Flt3L and that the recombinant standard is compatible (some kits may specify which standard to use).
• Follow Manufacturer Instructions: Use the recommended reconstitution buffer and dilution scheme to prepare the standard curve.
• Validate in Your Assay: If you are developing your own ELISA, validate the recombinant protein as a standard by generating a standard curve and confirming linearity and reproducibility.

In summary, Recombinant Mouse Flt-3 Ligand is a suitable and widely used standard for quantification in ELISA assays targeting mouse Flt3L.

Applications of Recombinant Mouse Flt-3 Ligand in Published Research

Recombinant mouse Flt-3 ligand (FLT3L) has been validated across a diverse range of applications in published research, spanning immunology, hematology, and therapeutic development.

Hematopoietic Cell Development and Expansion

FLT3L functions as a potent stimulator of hematopoietic cell proliferation and differentiation. The protein stimulates cell proliferation in the BaF3 mouse pro-B cell line transfected with mouse Flt-3, with an ED₅₀ of 0.4-2.4 ng/mL. Additionally, FLT3L synergizes with multiple cytokines including IL-3, GM-CSF, and stem cell factor to promote mobilization and myeloid differentiation of hematopoietic stem cells. When combined with stem cell factor and thrombopoietin, FLT3L is commonly used to promote expansion of primitive hematopoietic cells in culture.

Dendritic Cell Generation and Maturation

One of the most well-established applications involves dendritic cell (DC) development. FLT3L controls the development of dendritic cells and is particularly important for plasmacytoid DCs and CD8-positive classical DCs and their CD103-positive tissue-resident counterparts. The protein induces maturation and expansion of dendritic cells both in vivo and for in vitro generation of bone marrow-derived DCs. Research has demonstrated that FLT3L therapy increases all classical dendritic cell subsets in orthotopic breast cancer and lung cancer models.

B and T Cell Development

FLT3L cooperates with IL-2, IL-6, IL-7, and IL-15 to induce natural killer cell development. The protein also works with IL-3, IL-7, and IL-11 to induce terminal B cell maturation. Furthermore, FLT3L facilitates formation of regulatory T cells (Tregs), which has been shown to reduce the severity of antigen-induced arthritis in mice.

Eosinophil Generation

FLT3L enables the generation of eosinophils from bone marrow progenitor cells, expanding its utility in studying myeloid lineage differentiation.

Inflammatory and Immunological Studies

The protein has been applied to studies on allergic airway inflammation in mouse models. Additionally, animal studies demonstrate that FLT3L reduces the severity of experimentally induced allergic inflammation. Published research shows that FLT3L treatment reduces enterovirus A71 lethality in mice with enhanced B cell responses, and acutely malnourished weanling mice administered FLT3L can support cell-mediated inflammatory responses.

Therapeutic and Bioassay Applications

FLT3L has been validated for functional assays, ELISA, Western blot, and immunohistochemistry applications. The protein has been successfully expressed in recombinant form and used as a specific vehicle to deliver cytotoxic drugs to FLT3-expressing acute myeloid leukemia cells, demonstrating its utility in targeted therapeutic development.

Skeletal Myogenesis

Emerging research has identified FLT3L signaling as a novel regulator of skeletal myogenesis, with studies demonstrating that FLT3L depletion impairs differentiation of mouse primary myoblasts.

To reconstitute and prepare Recombinant Mouse Flt-3 Ligand (FLT3L) protein for cell culture experiments, dissolve the lyophilized protein in sterile water or PBS at a concentration between 50–100 µg/mL. For optimal stability and activity, further dilute in cell culture medium or buffer containing at least 0.1% BSA to prevent adsorption and loss of bioactivity.

Step-by-step protocol:

• Before opening the vial: Briefly centrifuge to collect all lyophilized material at the bottom.
• Reconstitution:
  • Add sterile water or PBS to achieve the desired stock concentration (commonly 50–100 µg/mL).
  • Gently mix by pipetting or vortexing; avoid vigorous agitation or foaming.
  • Allow the solution to sit at room temperature for several minutes to ensure complete dissolution.
• Aliquoting and storage:
  • Prepare small aliquots to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • For long-term storage, keep aliquots at –80°C (short-term at –20°C is acceptable).
• Working solution preparation:
  • Dilute the stock solution into cell culture medium or buffer containing at least 0.1% BSA (bovine or human serum albumin) to minimize protein loss due to adsorption.
  • Typical working concentrations for cell culture assays range from 0.4–2.4 ng/mL for bioactivity, but optimal concentrations depend on your specific cell type and experimental design.
• General precautions:
  • Avoid multiple freeze-thaw cycles.
  • Use sterile technique throughout to prevent contamination.
  • Do not use lysis buffers containing harsh detergents (e.g., RIPA) if preparing for ELISA or bioassay.

Summary Table:

Key points:

• Use sterile water or PBS for initial reconstitution.
• Always include BSA in working solutions to stabilize the protein.
• Store aliquots at –80°C for long-term use.
• Adjust final concentration based on cell type and experimental needs.

These steps ensure maximal recovery, stability, and bioactivity of recombinant mouse Flt-3 Ligand for cell culture applications.