Recombinant Mouse Fas Ligand

Recombinant Mouse Fas Ligand (FasL) is widely used in research applications to study and manipulate apoptosis, immune regulation, and cell signaling due to its well-characterized role in inducing cell death via the Fas receptor pathway.

Key scientific reasons to use recombinant mouse FasL include:

• Induction of Apoptosis: FasL binds to the Fas receptor (CD95) on target cells, triggering the extrinsic apoptotic pathway through caspase-8 activation. This is essential for investigating programmed cell death mechanisms in various cell types, including lymphocytes, tumor cells, and stem cells.

• Immune System Studies: FasL-Fas interactions are critical for immune homeostasis, including the elimination of autoreactive or excess lymphocytes, and for maintaining immune privilege in certain tissues. Recombinant FasL allows precise control over these processes in vitro and in vivo, facilitating studies on autoimmunity, immune tolerance, and transplant rejection.

• Modeling Disease and Therapeutic Interventions: Recombinant FasL is used to model pathological conditions such as viral infections, autoimmune diseases, and cancer, where Fas-mediated apoptosis is dysregulated. It also enables testing of therapeutic strategies, such as immunosuppression or targeted cell depletion.

• Functional Assays and Cell Culture: Recombinant FasL is commonly used in cell-based assays to assess Fas sensitivity, apoptosis induction, and downstream signaling events. It serves as a standard for ELISA and other quantitative assays.

• Protein Engineering and Mechanistic Studies: The recombinant form allows for modifications (e.g., soluble vs. membrane-bound, tagged versions) to dissect structure-function relationships, receptor binding, and signaling specificity.

Best practices when using recombinant mouse FasL:

• Use appropriate controls, such as Fas-deficient cells, to confirm specificity of apoptosis induction.
• Select the correct form (soluble or membrane-bound) depending on the experimental context, as biological activity and receptor engagement may differ.
• Optimize concentration and exposure time for your cell type and assay, as ED50 values can vary.

In summary, recombinant mouse FasL is a powerful tool for dissecting apoptosis, immune regulation, and related signaling pathways in mouse models and cell systems, enabling mechanistic and translational research in immunology, cell biology, and disease modeling.

Yes, you can use recombinant mouse Fas Ligand as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately known. This is a common and accepted practice in ELISA development and quantification protocols.

Key considerations and supporting details:

• Recombinant protein standards are routinely used to generate standard curves in ELISA assays for quantification of the target analyte in biological samples. The standard curve allows interpolation of sample concentrations based on their signal relative to known concentrations of the recombinant standard.

• Purity and quantification: The recombinant Fas Ligand should be highly purified, and its concentration should be accurately determined (e.g., by absorbance at 280 nm, BCA assay, or HPLC). Impurities or inaccurate quantification can introduce error into your standard curve and subsequent sample measurements.

• Formulation: Some suppliers recommend using recombinant proteins with carrier proteins (such as BSA) for ELISA standards to improve stability and minimize adsorption to plasticware. Carrier-free forms are also available but may require additional precautions to prevent loss of protein.

• Matrix effects: The standard should be diluted in the same buffer or matrix as your samples to minimize matrix effects, which can alter the apparent concentration due to differences in protein binding or interference. Internal controls or spiked recovery experiments are recommended to validate assay accuracy in your specific sample matrix.

• Kit compatibility: If you are using a commercial ELISA kit, check the kit documentation to ensure compatibility with recombinant standards. Many kits are validated using recombinant mouse Fas Ligand as the standard.

• Standard curve preparation: Prepare a fresh standard curve for each assay run, following best practices for serial dilution and pipetting accuracy.

Summary of best practices:

• Use a highly purified, accurately quantified recombinant mouse Fas Ligand.
• Prepare standards in the same buffer/matrix as your samples.
• Validate recovery and linearity in your sample matrix.
• Follow the ELISA kit or protocol instructions for standard curve preparation.

This approach is widely used and supported by both general ELISA guidelines and specific protocols for mouse Fas Ligand quantification.

Recombinant Mouse Fas Ligand (FasL) has been validated for several key applications in published research, primarily centered on its role in inducing apoptosis, modulating immune responses, and studying cell signaling pathways.

Validated Applications in Published Research:

• Induction of Apoptosis in Fas-Expressing Cells:
  Recombinant mouse FasL is widely used to trigger apoptosis in vitro in Fas-expressing cell lines, such as T cells and fibroblasts, by binding to the Fas (CD95) receptor. This is a standard assay to study Fas-mediated cell death and downstream signaling.

• In Vivo Functional Studies:
  Recombinant mouse FasL has been administered to mice to study its effects on immune cell populations, tissue injury, and systemic toxicity. For example, it has been used to induce apoptosis in mouse models to investigate immune regulation and tissue-specific responses.

• Modulation of Immune Responses and Transplantation Models:
  FasL has been used in gene therapy and transplantation research to modulate immune responses. For instance, adenoviral vectors encoding mouse FasL have been employed to protect transduced cells from immune rejection and to study immune privilege in tissues such as the liver and muscle.

• Inflammation and Lung Injury Models:
  Soluble recombinant FasL has been used in mouse models of lung inflammation to assess its impact on inflammatory cell recruitment, cytokine production, and tissue injury, particularly in the context of acute lung injury or infection.

• ELISA and Binding Assays:
  Recombinant FasL is used as a standard or functional ligand in ELISA-based assays to measure binding interactions, such as the development of Fas decoy receptors or to quantify FasL in biological samples.

• Non-Apoptotic Signaling and Developmental Studies:
  Beyond apoptosis, recombinant FasL has been used to explore non-apoptotic roles, such as its involvement in osteoblast function and bone development, where it modulates gene expression independently of cell death.

Additional Notes:

• Bioactivity Validation:
  The bioactivity of recombinant mouse FasL is typically validated by its ability to induce apoptosis in sensitive cell lines (e.g., Jurkat T cells), with effective dose ranges reported in the literature.

• In Vitro and In Vivo Use:
  Applications span both in vitro cell culture systems and in vivo animal models, reflecting its utility in mechanistic studies and preclinical research.

• Controls and Specificity:
  Functional assays often include Fas-deficient cells or blocking antibodies to confirm specificity of FasL-induced effects.

Summary Table: Applications of Recombinant Mouse Fas Ligand in Research

These applications are well-supported in the literature and reflect the broad utility of recombinant mouse Fas Ligand in immunology, cell biology, and translational research.

To reconstitute and prepare Recombinant Mouse Fas Ligand protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) containing at least 0.1–1% human or bovine serum albumin (BSA), aiming for a final concentration of 100 μg/mL. Avoid vigorous mixing or vortexing, as this may impair biological activity.

Step-by-step protocol:

• Centrifuge the vial briefly to collect all lyophilized powder at the bottom before opening.
• Add sterile PBS (with 0.1–1% BSA) to achieve a concentration of 100 μg/mL.
• Gently mix by pipetting up and down or allowing the solution to sit at room temperature for at least 20 minutes to ensure complete dissolution. Do not vortex.
• Aliquot the stock solution to avoid repeated freeze-thaw cycles.
• Store the reconstituted protein at 2–8 °C for short-term use (up to 1 week), or at –20 °C or colder for longer-term storage.
• Dilute the stock to the desired working concentration in cell culture medium immediately before use. Typical effective concentrations for cell-based assays range from 0.4 to 1.2 μg/mL, but optimal conditions should be empirically determined for your specific application.

Additional notes:

• The inclusion of BSA or another carrier protein helps stabilize Fas Ligand and prevents adsorption to tube walls.
• If the product datasheet specifies a different buffer or carrier protein, follow those instructions.
• Always use sterile technique to prevent contamination.

Summary of key points:

• Buffer: Sterile PBS + 0.1–1% BSA
• Concentration: 100 μg/mL for stock
• Mixing: Gentle, no vortexing
• Storage: 2–8 °C (short-term), –20 °C (long-term)
• Working concentration: 0.4–1.2 μg/mL (empirical optimization recommended)

This protocol ensures optimal solubility and biological activity of recombinant mouse Fas Ligand for cell culture experiments.