Recombinant Human Flt-3

Recombinant Human Flt-3 Ligand (FLT3L) is widely used in research due to its critical role in the regulation, expansion, and differentiation of hematopoietic stem and progenitor cells, as well as its importance in immune cell development and disease modeling.

Key scientific applications and rationales include:

• Expansion of Hematopoietic Stem Cells: FLT3L is essential for the in vitro expansion and maintenance of primitive CD34⁺ hematopoietic progenitor cells, often in combination with other cytokines such as stem cell factor (SCF) and thrombopoietin.
• Differentiation of Immune Cells: It is crucial for the differentiation of dendritic cells in the myeloid lineage and for B cell and natural killer (NK) cell development in the lymphoid lineage.
• Synergistic Effects: FLT3L synergizes with cytokines like IL-3, GM-CSF, and SCF to promote myeloid differentiation, and with IL-2, IL-6, IL-7, and IL-15 to induce NK cell development.
• Disease Modeling and Functional Assays: Recombinant FLT3L is used in disease modeling (e.g., leukemia, aplastic anemia) and in functional assays to study hematopoietic and immune cell biology.
• High-Throughput Screening and Drug Development: Recombinant FLT3 and its ligands are used to assess kinase activity, screen for inhibitors, and study FLT3 signaling in the context of acute myeloid leukemia (AML) and other hematologic malignancies.
• Cell Culture and Differentiation Protocols: FLT3L is a standard component in protocols for generating dendritic cells, Langerhans cells, eosinophils, and other hematopoietic derivatives from stem or progenitor cells.

Summary of scientific benefits:

• Enables robust and reproducible expansion and differentiation of hematopoietic and immune cells.
• Facilitates mechanistic studies of FLT3 signaling in normal and malignant hematopoiesis.
• Supports translational research, including drug screening and disease modeling, particularly in leukemia and immune cell development.

Using recombinant human FLT3L ensures high purity, batch-to-batch consistency, and defined activity, which are essential for reproducible experimental outcomes in both basic and translational research.

Yes, recombinant human Flt-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the analyte detected by your assay. Recombinant Flt-3 Ligand is commonly used as a standard in commercial ELISA kits designed to quantify human Flt-3 Ligand in biological samples.

Key considerations for use as an ELISA standard:

• Validation: The recombinant Flt-3 Ligand must be validated for use as a standard in your specific ELISA format. Many commercial kits use recombinant Flt-3 Ligand as their calibration standard and demonstrate accurate quantification of both recombinant and natural forms.
• Formulation: Recombinant proteins formulated with carrier proteins (such as BSA) are generally recommended for use as ELISA standards due to increased stability and reduced adsorption to plasticware. Carrier-free formulations may be used if BSA interferes with your assay.
• Source and Sequence: Ensure the recombinant protein matches the sequence and post-translational modifications of the native analyte as closely as possible. Most commercial recombinant Flt-3 Ligand standards are produced in mammalian expression systems to ensure appropriate glycosylation.
• Concentration and Reconstitution: Follow manufacturer or protocol recommendations for reconstitution and dilution to prepare the standard curve. Typical standard curves for Flt-3 Ligand ELISA range from low pg/mL to several hundred pg/mL.
• Lot-to-Lot Consistency: High-quality recombinant standards should demonstrate consistent activity and quantification across lots, as verified by bioassay and analytical testing.

Protocol best practices:

• Prepare fresh standard dilutions for each assay to ensure accuracy.
• Use the same buffer and matrix for standard and sample dilutions to minimize matrix effects.
• Confirm that your ELISA kit or protocol is validated to detect both recombinant and endogenous Flt-3 Ligand, as most commercial kits are.

Limitations:

• Recombinant standards may differ from endogenous proteins in glycosylation or other modifications, which can affect antibody recognition in rare cases. Always verify compatibility with your specific ELISA antibodies and detection system.
• For bioassays (functional assays), standards intended for ELISA quantification may not be suitable unless specifically tested for biological activity.

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

In conclusion, recombinant human Flt-3 Ligand is appropriate for use as a standard in ELISA quantification and calibration, provided it is validated for your assay and prepared according to best practices.

Recombinant Human Flt-3 Ligand (FLT3L) has been validated in published research for several key applications, primarily in hematopoietic cell biology, immunology, and leukemia studies.

Validated Applications:

• Expansion and Differentiation of Hematopoietic Stem and Progenitor Cells:

  • Used to generate and expand CD34⁺ hematopoietic progenitor cells in vitro.
  • Facilitates differentiation of embryonic stem (ES) cell-derived progenitors toward the hematopoietic lineage.
  • Promotes maturation of neutrophils, eosinophils, and other myeloid cells from CD34⁺ sources.

• Dendritic Cell Generation and Function:

  • Critical for the in vitro generation of conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) from hematopoietic progenitors.
  • Used in studies of dendritic cell homeostasis and adaptive immunity, including infection and inflammation models.

• B Cell and NK Cell Development:

  • Cooperates with other cytokines (e.g., IL-2, IL-6, IL-7, IL-15) to induce NK cell development and terminal B cell maturation.

• Leukemia and Drug Development:

  • Validated in acute myeloid leukemia (AML) models for targeted drug delivery, such as ligand-drug conjugates (e.g., FL-Fc-DM1) that exploit FLT3 expression for selective cytotoxicity.
  • Used in studies of chemotherapy resistance, leukemia stem cell biology, and apoptosis induction in AML cells.

• Cell Culture and Bioassays:

  • Widely used as a supplement in cell culture media for functional assays, lineage differentiation, and expansion protocols.
  • Applied in bioassays to assess cytokine activity, cell proliferation, and lineage-specific differentiation.

• Disease Modeling and Immunology:

  • Utilized in disease modeling with induced pluripotent stem cells (iPSCs) for genetic diseases affecting hematopoiesis.
  • Studied in the context of autoimmune diseases (e.g., rheumatoid arthritis) and infection models (e.g., Plasmodium).

Summary Table of Applications

Additional Notes:

• FLT3L is often used in combination with other cytokines (e.g., SCF, IL-3, GM-CSF, thrombopoietin) to optimize hematopoietic cell expansion and differentiation protocols.
• Its role in dendritic cell biology makes it valuable for studies of antigen presentation, vaccine development, and immune regulation.
• In leukemia research, FLT3L-based constructs are being explored for targeted therapies and overcoming drug resistance.

These applications are supported by peer-reviewed studies and are widely adopted in both basic and translational research settings.

Reconstitution Protocol

Initial Preparation

Begin by centrifuging the vial briefly before opening to bring the lyophilized powder to the bottom of the tube. This prevents the powder from adhering to the tube walls or cap during handling. Allow both the vial and reconstitution buffer to equilibrate to room temperature before proceeding.

Buffer Selection and Reconstitution

Reconstitute the lyophilized protein in sterile phosphate-buffered saline (PBS) containing at least 0.1% bovine serum albumin (BSA) or human serum albumin as a carrier protein. The specific reconstitution concentration depends on your vial size: reconstitute 5 µg vials at 50 µg/mL, while 25 µg or larger vials should be reconstituted at 100 µg/mL in sterile PBS.

Mixing Technique

Add the buffer by gently pipetting the solution down the sides of the vial rather than directly onto the powder. Do not vortex, as vigorous agitation can denature the protein. Allow 15-30 minutes for gentle agitation to facilitate complete reconstitution. If flakes remain visible, continue mixing for approximately 2 hours at room temperature.

Storage Conditions

Short-term Storage

For experiments lasting up to one week, store the reconstituted protein at 2-8°C in a standard refrigerator. Under these conditions, the protein remains active for approximately one week, which is suitable for typical cell culture cycles of 5-7 days.

Long-term Storage

For extended storage or when the prepared protein cannot be used immediately, dilute the reconstituted protein with a carrier protein-containing solution (0.1% BSA, 10% fetal bovine serum, or 5% human serum albumin) and aliquot into working portions. Store these aliquots at -20°C to -80°C in a manual defrost freezer. Under these conditions, the protein remains stable for up to six months.

Critical Storage Considerations

Avoid repeated freeze-thaw cycles, as these significantly reduce protein stability and bioactivity. Use a manual defrost freezer rather than automatic defrost models to minimize temperature fluctuations. For serum-free culture applications or in vivo experiments, use trehalose as an alternative carrier protein instead of animal-derived proteins.

Quality Specifications

The recombinant protein typically exhibits purity ≥97% as determined by SDS-PAGE analysis, with endotoxin levels ≤0.1 EU/µg. Biological activity is confirmed through cell proliferation assays, with expected specific activity ≥2.0 × 10⁵ units/mg and ED₅₀ values ≤5 ng/mL.