Recombinant Human Fas Ligand (FasL)

Recombinant Human Fas Ligand (FasL) is widely used in research due to its central role in regulating apoptosis, immune homeostasis, and immune privilege, making it a valuable tool for studying cell death, immune tolerance, and related pathologies.

Key reasons to use recombinant human FasL in research applications:

• Induction of Apoptosis: FasL binds to its receptor (Fas/CD95) on target cells, triggering the extrinsic apoptotic pathway. This is essential for studying mechanisms of programmed cell death in various cell types, including lymphocytes, tumor cells, and cells in immune-privileged sites.
• Immune Regulation: FasL-Fas interactions are critical for maintaining immune tolerance by eliminating autoreactive or excess lymphocytes, thus preventing autoimmunity and lymphoproliferative disorders.
• Modeling Immune Privilege: FasL expression contributes to immune privilege in tissues such as the eye, testis, and brain by inducing apoptosis of infiltrating immune cells, which can be modeled in vitro using recombinant FasL.
• Cancer and Tumor Immunology: FasL is involved in tumor immune evasion and the elimination of transformed cells by cytotoxic T lymphocytes and NK cells. Recombinant FasL is used to study tumor cell sensitivity to immune-mediated apoptosis and to investigate therapeutic strategies targeting the Fas pathway.
• Transplantation and Tolerance: High FasL expression is associated with successful transplantation outcomes, such as corneal grafts, by promoting apoptosis of infiltrating immune cells and reducing rejection.
• Inflammation and Infection: FasL-mediated apoptosis regulates the resolution of immune responses and the elimination of pathogen-infected cells. Dysregulation can contribute to chronic inflammation or immunopathology, which can be modeled using recombinant FasL.
• Drug Screening and Mechanistic Studies: Recombinant FasL is used in bioassays to screen for modulators of apoptosis, to dissect signaling pathways, and to evaluate the effects of drugs or genetic modifications on Fas-mediated cell death.

Technical advantages of using recombinant FasL include:

• Defined, consistent activity and purity for reproducible results.
• Availability in soluble or membrane-bound forms to mimic physiological or experimental conditions.
• Suitability for a wide range of applications, including cell-based assays, mechanistic studies, and therapeutic research.

In summary, recombinant human FasL is a versatile reagent for investigating apoptosis, immune regulation, cancer biology, transplantation tolerance, and inflammatory responses, providing mechanistic insights and supporting the development of novel therapeutic strategies.

Recombinant Human Fas Ligand (FasL) can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the assay’s requirements.

Key considerations and supporting details:

• ELISA kits for human Fas Ligand commonly use recombinant FasL as the standard. These kits are designed to quantify both natural and recombinant FasL in biological samples, and the standard curves are generated using recombinant protein. For example, a widely used ELISA kit states: “It contains CHO cell-expressed recombinant human Fas Ligand and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Fas Ligand showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards”. This demonstrates that recombinant FasL is suitable as a standard for quantification.

• The recombinant standard must be prepared and used according to the assay protocol. ELISA protocols specify reconstitution, dilution, and storage conditions for the recombinant standard to ensure accuracy and reproducibility. For example, standards are typically serially diluted to generate a standard curve covering the assay’s dynamic range.

• The recombinant FasL used as a standard should be of high purity and well-characterized. It should match the isoform and post-translational modifications (if relevant) of the analyte being measured, as differences can affect antibody recognition and quantification accuracy. Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to enhance stability, which is generally acceptable for use as ELISA standards.

• Validation is essential. If you are using a recombinant FasL preparation not supplied with your ELISA kit, you must validate that it produces a standard curve comparable to the kit’s original standard and that it is recognized equivalently by the assay antibodies. Some manufacturers note that not all recombinant proteins are validated for use as ELISA standards in every kit, so cross-validation is required.

• Do not use a standard curve from a previous run. A fresh standard curve must be generated for each assay to account for potential variability in assay conditions and standard preparation.

Summary Table: Use of Recombinant FasL as ELISA Standard

In conclusion:
You can use recombinant human Fas Ligand as a standard for ELISA quantification if it is validated for your assay and prepared according to the protocol. Always ensure the recombinant protein is recognized by the assay antibodies and generates a standard curve comparable to the kit’s original standard.

Recombinant Human Fas Ligand (FasL) has been validated for several key applications in published research, primarily related to its role in inducing apoptosis and studying immune regulation.

Validated Applications in Published Research:

• Bioassays (Functional Assays):

  • Widely used to induce apoptosis in various cell types, especially Jurkat T cells and other lymphocytes, to study cell death mechanisms and immune regulation.
  • Used to assess sensitivity of cells (e.g., cancer cells, B cells) to Fas-mediated apoptosis, often in the context of cancer, autoimmune disease, or infection.
  • Applied in studies investigating the modulation of apoptosis by other factors, such as drugs or cytokines.

• Blocking Assays:

  • Used to block Fas-FasL interactions to study the consequences of inhibiting apoptosis pathways, particularly in immune and cancer research.

• Surface Plasmon Resonance (SPR):

  • Employed to characterize the binding kinetics and affinity between FasL and its receptor or other interacting molecules.

• In Vivo Functional Studies:

  • Utilized in animal models to investigate the therapeutic potential of FasL or FasL-fusion proteins for targeted induction of apoptosis in tumors or specific immune cell populations.
  • Demonstrated efficacy in reducing tumor growth and metastasis in xenograft models by inducing apoptosis in Fas-expressing tumor cells.

• Development of Therapeutic Proteins and Diagnostic Tools:

  • Recombinant FasL has served as a basis for engineering fusion proteins (e.g., antibody-FasL constructs) for targeted cancer therapy and for diagnostic assays targeting Fas/FasL pathway components.

Additional Context:

• Mechanistic Studies: Recombinant FasL is a standard tool for dissecting the molecular mechanisms of Fas-mediated apoptosis, including caspase activation and downstream signaling.
• Immunological Research: Used to study immune privilege, immune cell homeostasis, and the role of FasL in autoimmune diseases and transplantation tolerance.
• Protein Engineering: Modified forms of recombinant FasL (e.g., site-specific chemical modifications, fusion proteins) are validated for enhanced stability, targeting, or activity in both research and preclinical therapeutic development.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the broad utility of recombinant human FasL in both basic and translational research.

To reconstitute and prepare Recombinant Human Fas Ligand (FasL) protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS containing at least 0.1–1% human or bovine serum albumin (BSA or HSA) to a stock concentration of at least 10–100 μg/mL. This prevents adsorption to plastic and stabilizes the protein.

Step-by-step protocol:

• Reconstitution:

  • Add sterile PBS with 0.1–1% BSA or HSA to the vial to reach your desired stock concentration (commonly 100 μg/mL, but not less than 10 μg/mL).
  • Gently pipette up and down or swirl to dissolve. Avoid vigorous vortexing to prevent protein denaturation.

• Aliquoting and Storage:

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20 °C or –70 °C for long-term storage; at 2–8 °C for up to one month.
  • Avoid repeated freeze-thaw cycles, as this can reduce activity.

• Working Solution Preparation:

  • For cell culture, dilute the stock solution to the desired working concentration using cell culture medium or PBS containing at least 0.1% carrier protein (BSA, HSA, or 5% fetal calf serum).
  • Typical working concentrations for functional assays are in the ng/mL to low μg/mL range, depending on the assay and cell type.

Additional notes:

• If the protein is supplied without carrier protein, always add BSA or HSA during reconstitution to minimize loss from adsorption.
• For bioactivity, some applications may require cross-linking with an anti-FasL antibody to enhance Fas receptor activation.
• Always consult the specific product datasheet for any unique instructions.

Summary Table:

This protocol ensures protein stability and bioactivity for cell culture experiments.