Recombinant Human Fms-related tyrosine kinase 3 ligand (FLT3LG) Ligand

Recombinant Human Fms-related tyrosine kinase 3 ligand (FLT3LG) is widely used in research for its critical roles in hematopoiesis, immune cell development, and modulation of antitumor immune responses. Its application is particularly valuable in studies involving dendritic cell biology, hematopoietic stem cell expansion, and cancer immunotherapy.

Key scientific reasons to use recombinant FLT3LG in research applications:

• Expansion and Differentiation of Hematopoietic Progenitors: FLT3LG is a potent stimulator of primitive and committed human CD34+ bone marrow progenitor cells, promoting their proliferation and differentiation into various lineages, including dendritic cells (DCs), monocytes, and B cells.

• Dendritic Cell Generation: FLT3LG is essential for the differentiation and expansion of both conventional and plasmacytoid dendritic cells from FLT3+ bone marrow progenitors, making it a standard reagent for generating DCs in vitro for immunological studies.

• Synergy with Other Cytokines: FLT3LG 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 hematopoietic and immune cells, including NK cells and B cells.

• Immune Modulation and Antitumor Activity: Elevated FLT3LG expression is associated with increased infiltration and activation of immune cells (e.g., T cells, NK cells) in tumors, improved prognosis in certain cancers, and enhanced efficacy of immunotherapies such as anti-PD-1 checkpoint inhibitors. FLT3LG can be used to study mechanisms of immune cell recruitment and activation in the tumor microenvironment.

• Biomarker and Prognostic Value: FLT3LG levels can serve as diagnostic and prognostic markers in various malignancies, including lung adenocarcinoma and acute myeloid leukemia (AML), and may predict responsiveness to immunotherapeutic interventions.

• Hematopoietic Stem Cell Culture: In combination with other growth factors (e.g., stem cell factor, thrombopoietin), FLT3LG is commonly used to promote the expansion of primitive hematopoietic stem and progenitor cells in culture, supporting studies in stem cell biology and transplantation.

• Modeling Resistance Mechanisms: In leukemia research, FLT3LG is used to model resistance to FLT3 inhibitors and to study the impact of ligand-mediated signaling on drug efficacy.

In summary, recombinant FLT3LG is a versatile tool for research in immunology, hematology, oncology, and stem cell biology, enabling the controlled study of cell differentiation, immune modulation, and therapeutic responses in both basic and translational settings.

Yes, recombinant human FLT3 ligand (FLT3LG) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, accurately quantified, and compatible with the antibodies and detection system used in your assay.

Essential context and supporting details:

• ELISA Standard Requirements: For quantitative ELISA, the standard should be the same analyte as the target in your samples, ideally in recombinant form for consistency and reproducibility. Recombinant human FLT3LG is commonly used as a standard in commercial ELISA kits for human FLT3LG quantification.

• Purity and Activity: The recombinant protein should be highly pure (≥95% by SDS-PAGE is typical) and its concentration should be accurately determined, usually by absorbance at 280 nm or a validated protein assay. Activity is sometimes referenced to a WHO standard for calibration, ensuring traceability.

• Preparation and Dilution: Standards are typically reconstituted and serially diluted in the same buffer used for samples to generate a standard curve covering the assay’s dynamic range. For example, standard curves in FLT3LG ELISA kits often range from low pg/mL to several hundred pg/mL.

• Carrier Proteins: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to enhance stability. For ELISA calibration, carrier-free preparations are preferred if BSA or other additives could interfere with antibody binding or detection.

• Validation: It is recommended to validate the recombinant FLT3LG standard in your specific ELISA system, confirming parallelism between the standard curve and endogenous FLT3LG in your sample matrix. This ensures accurate quantification and recovery.

Best Practices:

• Use a recombinant FLT3LG standard that is traceable to an international reference (e.g., WHO standard) when possible for inter-laboratory comparability.
• Prepare fresh dilutions for each assay and avoid repeated freeze-thaw cycles to maintain protein integrity.
• Confirm that the recombinant standard matches the isoform and post-translational modifications (if relevant) of the FLT3LG detected by your ELISA antibodies.

Limitations:

• If your ELISA is designed for a different species (e.g., mouse FLT3LG), human recombinant FLT3LG may not be suitable due to potential differences in antibody recognition.
• Always verify compatibility with your specific ELISA kit or custom assay, as antibody specificity and epitope recognition can vary.

In summary, recombinant human FLT3LG is appropriate as a standard for ELISA quantification, provided it meets purity, quantification, and compatibility requirements for your assay system.

Recombinant Human Fms-related tyrosine kinase 3 ligand (FLT3LG) has been validated for use in several key applications in published research, including:

• Functional Assays: FLT3LG is used to study its biological activity, such as the expansion and differentiation of hematopoietic stem cells, dendritic cells, and immune cell subsets.
• ELISA (Enzyme-Linked Immunosorbent Assay): FLT3LG expression levels have been measured in serum and other biological samples using ELISA, for example, in studies of lung adenocarcinoma and other cancers.
• Western Blot: Detection of FLT3LG protein expression in cell lysates or tissue samples.
• Immunohistochemistry (IHC): Used to assess FLT3LG expression and localization in tumor tissues and to evaluate immune cell infiltration.
• Flow Cytometry: Employed to analyze immune cell populations and their activation status in response to FLT3LG, particularly in mouse models and in vitro studies.
• In Vivo Studies: FLT3LG has been used in animal models to investigate its effects on immune cell infiltration, tumor progression, and response to immunotherapy.

These applications highlight FLT3LG's role in immunology, oncology, and hematopoiesis research, supporting its use as a biomarker and therapeutic target.

To reconstitute and prepare Recombinant Human Fms-related tyrosine kinase 3 ligand (FLT3LG) protein for cell culture experiments, follow these general best practices based on manufacturer guidelines and scientific protocols:

Reconstitution

1. Centrifuge the vial: Briefly centrifuge the lyophilized protein vial before opening to ensure all contents are at the bottom.
2. Reconstitution buffer: Reconstitute the lyophilized protein in sterile, distilled water or an appropriate buffered solution (such as PBS or Tris buffer) containing 0.1% BSA (bovine serum albumin) or 0.1% endotoxin-free recombinant human serum albumin (HSA) to stabilize the protein and maintain full activity.
3. Concentration: Reconstitute to a concentration recommended by the manufacturer (commonly 0.1–0.2 mg/mL or as specified on the certificate of analysis).
4. Mix gently: Gently swirl or tap the vial to dissolve the protein. Avoid vigorous vortexing or pipetting, which can denature the protein.

Preparation for Cell Culture

1. Aliquot the reconstituted protein: Divide the reconstituted protein into working aliquots to minimize freeze-thaw cycles.
2. Storage: Store aliquots at ≤ -20°C for long-term use. For short-term use, the reconstituted protein can be stored at 4–8°C for up to 1 week.
3. Dilution for cell culture: When preparing working solutions for cell culture, dilute the reconstituted protein in cell culture medium or a buffered solution containing a carrier protein (e.g., PBS + 0.1% BSA).

Additional Notes

• Avoid repeated freeze-thaw cycles: Repeated freezing and thawing can reduce protein activity.
• Sterility: Ensure all steps are performed under aseptic conditions to prevent contamination.
• Carrier protein: The addition of BSA or HSA helps stabilize the protein and prevents adsorption to surfaces.

By following these steps, you can ensure the recombinant FLT3LG protein remains active and suitable for cell culture experiments.