Recombinant Mouse Flt-3

Recombinant Mouse Flt-3 Ligand (Flt3L) is widely used in research to promote the differentiation, proliferation, and expansion of hematopoietic progenitor cells, especially for generating and studying dendritic cells, early lymphoid and myeloid lineages, and for modeling immune responses in vitro and in vivo.

Key scientific applications and rationale include:

• Expansion and Differentiation of Hematopoietic Progenitors: Flt-3 Ligand directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells (Lin-Sca-1+), both alone and synergistically with other hematopoietic growth factors such as IL-3, GM-CSF, and SCF. This is essential for studies on stem cell biology, hematopoiesis, and immune cell development.

• Generation of Dendritic Cells: Flt-3L is a standard cytokine for inducing the maturation and expansion of dendritic cells (DCs) from mouse bone marrow progenitors, both in vitro and in vivo. This is critical for immunology research, vaccine development, and studies of antigen presentation.

• Lymphoid and Myeloid Lineage Studies: Flt-3L synergizes with cytokines such as IL-2, IL-6, IL-7, and IL-15 to promote NK cell development, and with IL-3, IL-7, and IL-11 for B cell maturation. This makes it valuable for dissecting pathways of lymphopoiesis and myelopoiesis.

• Functional Assays and Disease Models: Recombinant Flt-3L is used in functional assays to study immune responses, allergic inflammation, and hematological diseases in mouse models. It is also used to enhance the sensitivity of antigen-specific B and T cell responses.

• Cell Culture Supplementation: Flt-3L is commonly added to culture media to support the survival, proliferation, and differentiation of early hematopoietic cells, especially when combined with stem cell factor (SCF) and thrombopoietin (TPO).

• Synergy with Other Cytokines: Flt-3L’s ability to synergize with multiple cytokines allows for tailored manipulation of hematopoietic and immune cell populations for experimental needs.

In summary, using recombinant mouse Flt-3 Ligand is essential for any research requiring robust expansion or differentiation of hematopoietic progenitors, generation of dendritic cells, or modeling of immune cell development and function in murine systems.

Yes, you can use recombinant mouse Flt-3 ligand (Flt3L) as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common practice in quantitative ELISA protocols for cytokines and growth factors.

Key considerations and supporting details:

• Intended Use: Recombinant mouse Flt-3 ligand is routinely used as a standard in commercial ELISA kits designed to quantify Flt3L in biological samples. These kits typically include a lyophilized recombinant Flt3L standard, which is reconstituted and serially diluted to generate a standard curve for quantification.

• Purity and Activity: The recombinant protein should be of high purity (typically >85–95% by SDS-PAGE) and free from significant endotoxin contamination. The biological activity (e.g., EC50 or ED50) is sometimes provided, but for ELISA calibration, the critical factor is the accurate mass concentration, not bioactivity per se.

• Standard Curve Preparation: The standard must be reconstituted and diluted according to the ELISA kit or assay protocol to generate a standard curve covering the expected concentration range in your samples (e.g., 15–5000 pg/mL, depending on the kit). Each assay should include a freshly prepared standard curve.

• Compatibility: Ensure the recombinant Flt3L matches the species and isoform recognized by the antibodies in your ELISA. Most mouse Flt3L ELISAs are validated to detect both natural and recombinant forms.

• Documentation: If using a recombinant Flt3L not supplied with your ELISA kit, confirm that it is suitable as a standard by checking the kit documentation or contacting the kit provider. Some vendors note that their recombinant proteins are suitable for use as ELISA standards, while others caution that not all recombinant proteins are validated for this purpose.

• Critical Controls: Always run the standard curve in parallel with your samples in each assay to ensure accurate quantification.

Summary Table: Recombinant Mouse Flt-3 Ligand as ELISA Standard

In summary: Using recombinant mouse Flt-3 ligand as a standard is standard practice for quantitative ELISA, provided the protein is pure, accurately quantified, and compatible with your assay system. Always follow the specific instructions of your ELISA kit for optimal results.

Recombinant Mouse Flt-3 Ligand (FLT3L) has been validated for a wide range of applications in published research, primarily focusing on hematopoietic cell biology, immune cell differentiation, and disease modeling. Key applications include:

• Bioassay: Used to assess the biological activity of FLT3L in promoting hematopoietic cell differentiation and proliferation, including studies on hematopoietic stem cell mobilization and myeloid differentiation.
• Cell Culture: Employed to expand and differentiate hematopoietic progenitor cells, dendritic cells, and other immune cell types in vitro.
• Functional Assay: Validated for evaluating the functional effects of FLT3L on cell signaling, proliferation, and differentiation.
• ELISA: Used as a standard or in detection systems for quantifying FLT3L or related proteins.
• Western Blot: Applied for protein detection and characterization in research settings.
• Immunohistochemistry: Utilized for detecting FLT3L expression in tissue samples.
• Studies on Allergic Airway Inflammation: Used in mouse models to investigate the role of FLT3L in allergic responses.
• Dendritic Cell Generation: Applied for the in vitro and in vivo generation of dendritic cells from bone marrow progenitors.
• B Cell and T Cell Maturation Studies: Employed in research on terminal B cell maturation and antigen-specific B and T cell responses.
• Leukemia and Cancer Research: Used in models to study the effects of FLT3L on leukemia cell lines and in therapeutic targeting of FLT3-expressing cancers.

These applications highlight the versatility of Recombinant Mouse Flt-3 Ligand in both basic research and translational studies.

To reconstitute and prepare Recombinant Mouse Flt-3 Ligand (Flt3L) protein for cell culture experiments, dissolve the lyophilized protein at 50 μg/mL in sterile PBS. For optimal stability and activity, include 0.1%–1% carrier protein such as bovine serum albumin (BSA) or human serum albumin (HSA) in the buffer.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile PBS (phosphate-buffered saline) to achieve a final concentration of 50 μg/mL. For example, add 1 mL PBS to a vial containing 50 μg protein.
• Include carrier protein: Add BSA or HSA to a final concentration of 0.1%–1% to minimize adsorption and stabilize the protein, especially for long-term storage or repeated freeze-thaw cycles.
• Gently mix by pipetting up and down or vortexing briefly. Avoid vigorous agitation or foaming.
• Allow the solution to sit at room temperature for 5–10 minutes to ensure complete dissolution.
• Aliquot the reconstituted protein into working volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at −20°C to −80°C for long-term use. For short-term use (up to several days), store at 2–8°C.

Preparation for cell culture:

• Dilute the stock solution to the desired working concentration in cell culture medium immediately before use. Typical effective concentrations for bioassays range from 0.4–2.4 ng/mL for stimulation of hematopoietic cells.
• Avoid repeated freeze-thaw cycles to preserve protein activity.
• Sterility: Ensure all buffers and solutions are sterile to prevent contamination in cell culture.

Additional notes:

• If the protein is supplied with BSA as a carrier, you may omit additional carrier protein unless higher stability is required.
• Always consult the specific Certificate of Analysis or product datasheet for details on formulation and recommended reconstitution buffer, as some preparations may differ slightly.
• For functional assays, confirm activity using a proliferation assay (e.g., BaF3-Flt3 cells) if needed.

Summary Table:

This protocol ensures maximal stability and bioactivity of recombinant mouse Flt-3 ligand for cell culture experiments.