FAS Antibody (CD95)

What is FAS (CD95) and what are its primary functions in cellular processes?

FAS (CD95, APO-1) is a 46 kDa transmembrane glycoprotein that functions as a cell death receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily . It plays a crucial role in programmed cell death through the extrinsic apoptotic pathway. When FAS interacts with its ligand (FasL) or agonistic antibodies, it triggers the aggregation of its intracellular FAS-associated death domains (FADD), leading to the formation of the death-inducing signaling complex (DISC) and subsequent activation of caspases . This cascade ultimately results in apoptosis.

Beyond apoptosis, FAS activates additional signaling pathways including NF-kappaB and MAPK3/ERK1 . In the immune system, FAS-mediated apoptosis contributes to peripheral tolerance and the antigen-stimulated suicide of mature T-cells, which is critical for maintaining immune homeostasis .

Which cell types express FAS (CD95) and how does expression vary across tissues?

FAS (CD95) is expressed by a broad range of both hematopoietic and non-hematopoietic cells. Among hematopoietic cells, it is found on monocytes, neutrophils, and lymphocytes . In non-hematopoietic tissues, FAS is expressed on fibroblasts and various other cell types .

Expression levels vary significantly depending on cell activation status, differentiation stage, and tissue microenvironment. In cancer research, altered FAS expression is frequently observed, with many tumor cells showing reduced FAS expression as a potential mechanism to evade immune surveillance and apoptosis .

When designing experiments to study FAS, it's important to consider these expression variations and verify expression levels in your specific experimental system using flow cytometry or western blotting before proceeding with functional studies.

What are the different types of FAS antibodies available for research, and how should they be selected?

Several types of FAS antibodies are available for research applications, each with specific properties and use cases:

• Agonistic antibodies (like clone CH-11) that mimic FasL and can induce apoptosis in FAS-expressing cells

• Neutralizing antibodies that block FAS-FasL interaction

• Detection antibodies for techniques like flow cytometry, western blotting, and immunohistochemistry

• Conjugated antibodies with various fluorophores (e.g., CoraLite® Plus 405) for flow cytometry and imaging applications

Selection criteria should include:

• Experimental application (e.g., functional studies vs. detection)

• Required species reactivity (e.g., human vs. mouse)

• Clone characteristics (e.g., DX2 does not block binding of EOS9.1, another antibody specific for human CD95)

• Format needs (conjugated vs. unconjugated)

• Validation status for your specific application

For functional studies inducing apoptosis, select clones known to have agonistic properties. For detection only, any well-validated antibody for your application of interest should suffice.

How should FAS antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of FAS antibodies are crucial for maintaining their activity and specificity:

Always refer to the specific manufacturer's recommendations as storage conditions may vary between products and preparations.

What are the typical applications for FAS antibodies in basic research?

FAS antibodies find utility in numerous basic research applications:

• Flow Cytometry (FC): FAS antibodies can be used to detect and quantify FAS expression on cell surfaces. Pre-titrated antibodies (e.g., 5 μl per 10^6 cells or 5 μl per 100 μl whole blood) are available for convenient use .

• Western Blotting (WB): For detecting FAS protein in cell or tissue lysates, with the expected molecular weight typically observed between 38-45 kDa .

• Immunohistochemistry (IHC): To visualize FAS expression patterns in tissue sections .

• Immunofluorescence (IF): For subcellular localization of FAS in fixed cells .

• ELISA: For quantitative detection of FAS protein in solution .

• Apoptosis induction: Agonistic FAS antibodies can be used to trigger apoptosis in cell culture by mimicking FasL, making them valuable tools for studying death receptor signaling pathways .

When designing experiments, consider the validated applications for your specific antibody clone and follow recommended protocols and dilutions for optimal results.

How do agonistic FAS antibodies differ in their mechanism of action compared to FasL, and what are the implications for experimental design?

Agonistic FAS antibodies and natural FasL differ in several key aspects that can impact experimental outcomes:

• Epitope targeting: FasL binds to specific regions of FAS, particularly involving a positively charged residue epitope (PPCR) in cysteine-rich domain 2 (CRD2) . Different agonistic antibodies may target different epitopes, resulting in varying levels of receptor clustering and signaling efficiency. The DX2 clone, for instance, doesn't block binding of EOS9.1, indicating they recognize different epitopes .

• Receptor clustering dynamics: Natural FasL, especially membrane-bound FasL, induces robust receptor clustering. Antibodies vary in their ability to replicate this clustering based on their valency and epitope recognition. Research has identified that engagement of the PPCR in CRD2 of Fas is critical for optimal receptor clustering and signaling .

• Signal strength and kinetics: FasL typically induces stronger and more rapid apoptotic signaling compared to most agonistic antibodies. This difference may impact experimental timelines and sensitivity.

Implications for experimental design:

• When studying physiological processes, consider whether antibody-induced signaling accurately represents natural FasL signaling

• For apoptosis studies, calibrate antibody concentrations and treatment durations carefully

• If receptor clustering is critical to your research question, select antibodies validated for efficient clustering

• Include appropriate controls, such as comparing antibody effects with recombinant FasL

• Consider microenvironmental factors that might influence antibody access to receptors versus natural ligand interactions

What molecular mechanisms contribute to resistance against FAS antibody-induced apoptosis in cancer cells, and how can these be experimentally addressed?

Cancer cells employ multiple mechanisms to evade FAS-mediated apoptosis, which researchers can investigate using various experimental approaches:

• Reduced FAS expression: Many resistant cancer cells downregulate FAS expression . This can be experimentally addressed by:

  • Quantifying FAS surface expression using flow cytometry

  • Measuring FAS mRNA and protein levels using qPCR and western blotting

  • Using epigenetic modifiers to determine if silencing is due to epigenetic mechanisms

  • Forced FAS re-expression to test if sensitivity can be restored

• Defective DISC formation: Impaired caspase-8 activation is observed in resistant cells . Experimental approaches include:

  • Immunoprecipitation of the DISC complex followed by western blotting to analyze component recruitment

  • Caspase-8 activity assays using fluorogenic substrates

  • Single-cell imaging of DISC assembly using fluorescently tagged components

• Altered expression of anti-apoptotic proteins: Increased expression of proteins like FLIP-L can inhibit apoptosis . Research strategies include:

  • Comparative protein expression analysis between sensitive and resistant cells (resistant U937 cells show 2.7-fold higher FLIP-L levels compared to wild-type)

  • siRNA knockdown or CRISPR knockout of candidate anti-apoptotic proteins

  • Pharmacological inhibition of anti-apoptotic proteins to test for sensitization

• Signaling pathway alterations: Resistance mechanisms involve altered tyrosine phosphatase/kinase activities and dependence on de novo protein synthesis . These can be studied by:

  • Phosphoproteomic analysis to identify differential signaling

  • Using inhibitors of protein synthesis (e.g., cycloheximide) or kinase/phosphatase activities

  • Pathway-specific reporter assays to monitor activity changes

• Cross-resistance mechanisms: Fas-resistant cells often show partial cross-resistance to other death receptor ligands like TRAIL and TNF-α , suggesting shared resistance mechanisms.

What are the critical considerations for optimizing FAS antibody-based apoptosis assays in primary cells versus cell lines?

Optimizing FAS antibody-based apoptosis assays requires different approaches for primary cells compared to established cell lines:

Primary Cells:

• Intrinsic variability: Primary cells from different donors exhibit variable FAS expression and sensitivity. Include multiple donors to account for biological variation.

• Culture adaptation effects: Primary cells may alter FAS expression and signaling with extended culture. Use cells at consistent, early passage numbers and verify FAS expression before experiments.

• Microenvironment dependency: Primary cells often require specific growth factors or cell-cell interactions that influence FAS sensitivity. Consider co-culture systems or conditioned media when relevant.

• Activation state: For immune cells, activation status dramatically affects FAS expression and sensitivity. Standardize activation protocols and document activation markers.

• Tissue-specific responses: Primary cells from different tissues respond differently to FAS stimulation. Adjust antibody concentrations and incubation times for each cell type.

Cell Lines:

• Clonal evolution: Continuous passaging can lead to clonal selection and altered FAS sensitivity. Use low-passage stocks and regularly validate line identity.

• Density-dependent effects: Many cell lines show density-dependent FAS sensitivity. Standardize seeding density and confluence at treatment time.

• Authentication: Verify cell line identity using STR profiling to ensure reproducibility.

Common Optimization Parameters:

• Antibody concentration: Titrate antibody concentrations (typically starting at ≤1 μg per test for flow cytometry) to determine optimal dose-response.

• Cross-linking requirements: Some FAS antibodies require cross-linking for optimal activity. Test with and without protein A/G or secondary antibodies.

• Incubation time: Apoptosis kinetics vary by cell type. Establish appropriate time courses (typically 4-24 hours).

• Detection methods: Combine multiple apoptosis detection methods for comprehensive assessment.

How does the structural binding of different FAS antibody clones affect downstream signaling and experimental outcomes?

The structural binding characteristics of different FAS antibody clones significantly impact downstream signaling and experimental outcomes:

• Epitope specificity and receptor conformation:

  • Different clones bind distinct epitopes across the FAS extracellular domains, with particular importance for the cysteine-rich domains (CRD1-3) .

  • A positively charged residue epitope (PPCR) in CRD2 has been identified as critical for optimal receptor clustering and signaling .

  • Antibodies targeting this PPCR region may induce superior FAS agonist signaling compared to those binding alternative epitopes.

  • The DX2 clone binds an epitope that doesn't interfere with EOS9.1 binding, indicating distinct binding sites with potentially different functional outcomes .

• Receptor clustering efficiency:

  • Higher-order clustering of FAS is essential for DISC assembly and apoptotic signaling .

  • Different antibody clones vary in their ability to promote receptor clustering.

  • Antibodies that effectively engage the PPCR epitope may induce more efficient clustering and consequently stronger signaling .

  • The valency of the antibody (bivalent IgG versus multivalent formats) further influences clustering dynamics.

• Signaling pathway activation profiles:

  • Beyond the canonical apoptotic pathway, FAS can activate multiple signaling cascades including NF-kappaB and MAPK pathways .

  • Different antibody clones may preferentially activate specific downstream pathways based on the receptor conformation they induce.

  • This selective pathway activation can result in varied biological outcomes from apoptosis to inflammation or even proliferation.

For experimental design, researchers should select clones with documented functional properties aligned with their research goals and consider using multiple clones to comprehensively investigate complex signaling outcomes.

What role does the FAS/CD95 system play in CAR-T cell therapy, and how can FAS antibodies be used to study these interactions?

The FAS/CD95 system has emerged as an important mediator in CAR-T cell therapy, particularly in relation to bystander killing effects:

How can researchers differentiate between soluble and membrane-bound forms of FAS in experimental systems?

FAS exists in both membrane-bound (approximately 46 kDa) and soluble (approximately 26 kDa) forms , which have distinct biological functions:

• Detection methods for distinguishing forms:

  • Western blotting: Membrane-bound FAS appears as a 38-45 kDa band, while soluble FAS appears at approximately 26 kDa .

  • Flow cytometry: Surface staining detects only membrane-bound FAS, while permeabilization allows detection of both forms.

  • ELISA: Specialized assays can be developed to specifically detect soluble FAS in culture supernatants or biological fluids.

• Isolation strategies:

  • Ultracentrifugation can separate membrane-bound (pellet) from soluble (supernatant) forms.

  • Immunoprecipitation with epitope-specific antibodies may preferentially capture one form over another.

  • Fractionation techniques can separate membrane components from cytosolic and secreted proteins.

• Functional characterization:

  • Membrane-bound FAS primarily mediates apoptotic signaling when engaged by FasL or agonistic antibodies.

  • Soluble FAS can act as a decoy receptor, potentially inhibiting FasL-mediated apoptosis.

  • Differential antibody binding assays can help determine the functional state of each form.

• Experimental considerations:

  • Cell culture conditions can affect the ratio of soluble to membrane-bound FAS through altered proteolytic processing.

  • Certain cell types preferentially produce soluble FAS, which should be considered when selecting experimental models.

  • Quantification of both forms provides important context for interpreting functional outcomes in FAS signaling studies.

• Methodological approaches:

  • Pulse-chase experiments can track the conversion of membrane-bound to soluble forms.

  • Proteolytic inhibitors can be used to determine if soluble FAS arises from enzymatic cleavage of the membrane form.

  • Expression systems with tagged versions of FAS can help monitor trafficking and processing in real-time.

Understanding the balance between these forms is crucial for interpreting experimental results and developing targeted therapeutic strategies involving the FAS/FasL system.