Recombinant Human Flt-3 Ligand

Recombinant Human Flt-3 Ligand is widely used in research applications to promote the expansion, differentiation, and maintenance of hematopoietic stem and progenitor cells, as well as to generate specific immune cell subsets such as dendritic cells, B cells, and natural killer (NK) cells.

Key scientific reasons to use recombinant human Flt-3 Ligand include:

• Expansion of Hematopoietic Stem and Progenitor Cells: Flt-3 Ligand is essential for the in vitro expansion of CD34⁺ hematopoietic progenitor cells, which are critical for studies involving stem cell biology, transplantation, and regenerative medicine.

• Differentiation of Immune Cell Lineages: It drives the differentiation of hematopoietic progenitors into myeloid and lymphoid lineages, notably supporting the development of dendritic cells (key antigen-presenting cells), B cells, and NK cells.

• Synergistic Effects with Other Cytokines: Flt-3 Ligand acts synergistically with cytokines such as IL-3, GM-CSF, SCF, IL-2, IL-6, IL-7, and IL-15 to enhance the mobilization, differentiation, and maturation of various immune cells.

• Functional Immunology Studies: It is used to generate dendritic cells for antigen presentation assays, vaccine development, and studies of innate and adaptive immunity.

• Disease Modeling and Therapeutic Research: Flt-3 Ligand is employed in disease models (e.g., leukemia, immunodeficiency, infection) to study hematopoietic and immune cell dynamics, and has shown protective effects in infection models by expanding dendritic cells and promoting Th1-type immune responses.

• Stem Cell Differentiation Protocols: It is a standard component in protocols for differentiating embryonic stem cells or induced pluripotent stem cells toward hematopoietic and immune lineages.

• Bioassays and Cell Culture: Recombinant Flt-3 Ligand is validated for use in cell culture systems, bioassays, and functional studies requiring the activation or proliferation of Flt-3 receptor-expressing cells.

In summary, recombinant human Flt-3 Ligand is a critical tool for hematopoietic and immunological research, enabling precise control over stem cell expansion, immune cell differentiation, and functional assays relevant to both basic science and translational applications.

Yes, recombinant human Flt-3 Ligand can be used as a standard for quantification or calibration in ELISA assays, provided it is of appropriate purity and validated for this purpose. Multiple ELISA kits for human Flt-3 Ligand specifically use recombinant protein as the standard for generating calibration curves.

Key considerations:

• Validation: ELISA kits designed to quantify human Flt-3 Ligand typically use recombinant Flt-3 Ligand as the standard, and these kits are validated to ensure that the recombinant standard produces a dose-response curve comparable to that of the natural protein.
• Purity and Form: The recombinant protein should be of high purity, and ideally, its sequence and post-translational modifications should match the native protein as closely as possible. Carrier-free or BSA-containing forms may be chosen depending on whether BSA interferes with your assay.
• Calibration: The recombinant standard is reconstituted and serially diluted to generate a standard curve, which is then used to quantify Flt-3 Ligand concentrations in unknown samples.
• Lot-to-lot consistency: High-quality recombinant standards are calibrated against international reference standards (e.g., NIBSC 96/532) to ensure consistency.

Best practices:

• Use a freshly prepared standard curve for each assay run to account for potential degradation or handling variability.
• Confirm that the recombinant standard is compatible with your specific ELISA kit and detection antibodies, as some kits are optimized for specific isoforms or glycosylation patterns.
• If using a recombinant standard not supplied with your ELISA kit, verify its equivalence by comparing its dose-response to that of the kit’s recommended standard.

Summary Table: Use of Recombinant Flt-3 Ligand as ELISA Standard

In conclusion, recombinant human Flt-3 Ligand is suitable as a standard for ELISA quantification, provided it is validated for this use and matches the assay requirements.

Recombinant Human Flt-3 Ligand has been validated for a range of applications in published research, primarily in the fields of hematopoiesis, immunology, and cancer biology.

Validated Applications:

• Functional Assays: Used to assess biological activity, such as stimulating proliferation and differentiation of hematopoietic progenitor cells, dendritic cells, and NK cells.
• Cell Culture: Widely used to expand and differentiate hematopoietic stem and progenitor cells, including CD34+ cells, and to generate dendritic cells, Langerhans cells, eosinophils, and NK cells in vitro.
• Bioassays: Employed in studies to confirm FLT3 signaling activation, cell proliferation, and differentiation, often using cell lines such as OCI-AML3 and BaF3.
• ELISA: Used as a standard or analyte in enzyme-linked immunosorbent assays to quantify FLT3 ligand levels in biological samples.
• Western Blot: Utilized as a positive control or to validate antibody specificity in immunoblotting experiments.
• Immunohistochemistry: Applied to tissue sections to study FLT3 ligand expression and localization.
• Preclinical and Clinical Studies: Investigated as an immunomodulatory agent in cancer immunotherapy trials, including for breast cancer, melanoma, and prostate cancer, often in combination with other agents.

Key Research Applications:

• Expansion and Differentiation of Hematopoietic Cells: Promotes the proliferation and differentiation of early hematopoietic progenitors, including myeloid and lymphoid lineages.
• Dendritic Cell Generation: Essential for the development of conventional and plasmacytoid dendritic cells, both in vitro and in vivo.
• Synergy with Other Cytokines: Works with cytokines such as IL-3, GM-CSF, SCF, IL-2, IL-6, IL-7, and IL-15 to enhance hematopoietic and immune cell development.
• Disease Modeling and Drug Delivery: Used as a targeting moiety for drug conjugates in FLT3-expressing acute myeloid leukemia (AML) cells, and for disease modeling in stem cell research.
• Immunotherapy and Immune Modulation: Studied for its role in expanding dendritic cells and modulating immune responses in cancer and infectious disease models.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed research, demonstrating the versatility of recombinant human Flt-3 Ligand in basic and translational studies.

To reconstitute and prepare Recombinant Human Flt-3 Ligand for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial:
Briefly spin down the lyophilized protein vial before opening to ensure all material is at the bottom.

2. Reconstitution:

• Use sterile water or sterile PBS as the solvent.
• Common reconstitution concentrations are 0.1 mg/mL (100 μg/mL) or 0.2 mg/mL (200 μg/mL).
• For improved stability and to prevent adsorption, add 0.1% human or bovine serum albumin (HSA/BSA) or another carrier protein if your application allows.
• Gently pipette or swirl (do not vortex) to dissolve the protein completely.

Example protocol:

- Add sterile water or PBS (with 0.1% HSA/BSA) to the vial to achieve 0.1–0.2 mg/mL.- Gently swirl or tap to mix until fully dissolved.

3. Aliquot and Storage:

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C to –80°C for long-term storage (up to 6 months), or at 4°C for short-term use (up to 1 week).
• Avoid repeated freeze-thaw cycles to maintain activity.

4. Working Solution Preparation:

• Dilute the stock solution to the desired working concentration using cell culture medium or buffer just before use.
• Typical working concentrations for cell culture range from 10–300 ng/mL, depending on the cell type and experimental design.

5. Additional Notes:

• If the protein is carrier-free, extra care should be taken to avoid loss due to adsorption; always use low-protein-binding tubes and consider adding carrier protein if compatible with your assay.
• Confirm the absence of endotoxin if using in sensitive cell cultures.

Summary Table:

These steps will ensure optimal recovery, stability, and biological activity of recombinant human Flt-3 Ligand for cell culture applications.