CFLAR Antibody

What is CFLAR and what are its primary functions in cellular pathways?

CFLAR, also known as FLIP (FLICE-inhibitory protein), belongs to the peptidase C14A family and functions as a crucial link between cell survival and cell death pathways in mammalian cells . It acts as an inhibitor of TNFRSF6-mediated apoptosis, with a proteolytic fragment (p43) likely retained in the death-inducing signaling complex (DISC), thereby blocking further recruitment and processing of caspase-8 at the complex . Different isoforms of CFLAR have varying effects, with some inducing apoptosis and others reducing TNFRSF-triggered apoptosis . Unlike caspases, CFLAR lacks enzymatic activity despite structural similarities .

What are the common synonyms for CFLAR in scientific literature?

CFLAR is referenced by multiple names in scientific publications, which can complicate literature searches. Common synonyms include: CASH, FLIP, MRIT, CLARP, FLAME, cFLIP, Casper, FLAME1, c-FLIP, FLAME-1, I-FLICE, c-FLIPL, c-FLIPR, c-FLIPS, and CASP8AP1 . When conducting literature reviews on CFLAR, researchers should include these alternative names in their search strategy to ensure comprehensive results.

What criteria should be considered when selecting a CFLAR antibody for research applications?

When selecting a CFLAR antibody, researchers should consider:

What are the recommended dilutions for CFLAR antibodies across different applications?

Optimal antibody dilutions vary by application and specific antibody preparation. Recommended ranges based on compiled data include:

It is essential to titrate antibodies for each specific experimental system to obtain optimal results .

What positive control samples are recommended for validating CFLAR antibody performance?

For rigorous validation of CFLAR antibody performance, the following positive controls are recommended:

When analyzing new sample types, including these validated positive controls in parallel experiments provides crucial reference points for antibody performance assessment .

What controls are essential for flow cytometry experiments using CFLAR antibodies?

For flow cytometry experiments with CFLAR antibodies, the following controls are critical:

• Single-stain controls: Must be run every time you perform an experiment, as variations in antibody staining, fluorophore stability, and instrument settings can affect results .

• Fluorescence Minus One (FMO) controls: Preferred over isotype controls for accurate gating as they account for spreading error from other fluorophores in the panel .

• Compensation beads vs. cells: Consider that compensation beads may not perfectly match cellular fluorophore behavior, particularly with polymer dyes (BUV, BV, BB, Super Bright) . When possible, use the same cell type for compensation as in your experiment.

• Unstained control: Essential for determining autofluorescence levels, particularly important for cell types like alveolar macrophages that exhibit high autofluorescence .

• Knockdown/knockout controls: Where available, samples with reduced or eliminated CFLAR expression provide definitive negative controls .

Proper labeling of all parameters and tubes with descriptive information (CD3-FITC, CD19-PE, etc.) is also important for accurate data interpretation and future reference .

What cell fixation and permeabilization protocols are recommended for CFLAR detection?

For optimal CFLAR detection in fixed cells, consider these protocol guidelines:

For immunofluorescence/immunocytochemistry:

• Fixation: Standard protocols using 4% paraformaldehyde are generally effective .

• Permeabilization: Use 0.1% Triton X-100 or similar detergent to allow antibody access to intracellular CFLAR.

• Blocking: Implement effective blocking (e.g., 5% BSA or 10% normal serum) to reduce background.

• Antibody incubation: Apply primary CFLAR antibody at appropriate dilution (typically 1:200-1:800) .

• Detection: For multiplex imaging, select compatible secondary antibodies such as "CY3-conjugated Goat Anti-Mouse IgG or Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG" .

• Counterstaining: DAPI nuclear counterstain provides cellular context for CFLAR localization .

For flow cytometry:

• Use commercial fixation/permeabilization kits compatible with intracellular proteins.

• Optimize fixation time to maintain epitope integrity while ensuring adequate permeabilization.

• Consider compensation requirements when selecting fluorophores, as CFLAR may be co-stained with other markers .

How can CFLAR antibodies be utilized to study the role of CFLAR cleavage in cell death regulation?

CFLAR cleavage plays a critical role in controlling cell death responses during tissue stress . Researchers can utilize CFLAR antibodies to study this process through:

• Detecting cleavage products: Use Western blotting with CFLAR antibodies to identify both full-length CFLAR (50-55 kDa) and cleaved fragments like p43 that remain in the DISC complex . This allows quantification of cleavage efficiency under different conditions.

• Mutation analysis: Compare wild-type samples with those expressing non-cleavable CFLAR mutants (e.g., D377A mutation) to understand functional consequences . CFLAR antibodies can confirm expression levels of mutant proteins.

• Complex formation studies: Use immunoprecipitation with CFLAR antibodies to isolate DISC complexes and analyze protein-protein interactions influencing cleavage .

• Tissue-specific analysis: Apply immunohistochemistry to examine CFLAR expression and cleavage patterns in tissues responding to stress, such as during viral infection or wound healing .

• Mechanistic investigations: Combine with analysis of glutamine-469 dependence, as research indicates this residue is crucial for the cell death-sensitizing effect of CFLAR cleavage inhibition .

This approach has revealed that abrogation of CFLAR cleavage sensitizes cells to TNF-induced necroptosis and apoptosis by favoring complex-II formation, with significant implications for tissue stress responses .

What insights have CFLAR expression studies provided in cancer research?

CFLAR expression studies in cancer have revealed several unexpected patterns:

• Downregulation in multiple cancers: Contrary to expectations, CFLAR expression is lower in breast cancer compared to normal tissue, with similar patterns observed in other cancer types .

• Inverse correlation with oncogenes: Analysis of TCGA data demonstrates an inverse relationship between oncogene expression (HRAS, AKT1) and CFLAR in breast and lung cancers . This suggests that cancer cells may benefit from lower CFLAR expression as a consequence of oncogenic signaling.

• Diagnostic potential: CFLAR has been identified as a novel diagnostic and prognostic biomarker in soft tissue sarcomas (STS) . Research methodologies including:

  • CIBERSORTx analysis to measure immune cell infiltration

  • Correlation between CFLAR expression and immune cell types

  • Single-sample gene set enrichment analysis to calculate immune function scores

  • TME scoring using the 'estimate' package to calculate 'StromalScore', 'ImmuneScore', and 'ESTIMATEScore'

• Single-cell analysis: Advanced studies have explored CFLAR expression at the single-cell level using technologies like Seurat for dimensionality reduction and cell clustering, revealing cell type-specific expression patterns .

These findings highlight the complex role of CFLAR in cancer biology and its potential as both a mechanistic target and biomarker.

How can CFLAR antibodies be incorporated into CAR-T cell research?

CFLAR has emerged as a potential target for chimeric antigen receptor (CAR) T-cell therapy, particularly for colorectal cancer . Research approaches include:

• Vector construction: Development of anti-CFLAR chimeric antigen receptor constructs, such as those using the scFv of anti-CFLAR antibody 7A3D12 linked to 4-1BB (CD137) and CD3ζ signaling domains .

• T-cell engineering: Genetic modification of T cells through transduction with lentiviral vectors expressing anti-CFLAR CAR constructs .

• Validation strategies:

  • Western blot to confirm CFLAR expression in target tissues

  • Flow cytometry to evaluate CAR expression on modified T cells

  • Cytotoxicity assays to assess CAR-T efficacy against CFLAR-expressing cells

  • Binding studies to determine CAR specificity using purified CFLAR protein

• In vivo models: Testing anti-CFLAR CAR-T cells in appropriate animal models of colorectal cancer to evaluate efficacy and safety profiles.

• Combination approaches: Investigating potential synergies between anti-CFLAR CAR-T cells and other immunotherapy or conventional treatment modalities.

This application represents an innovative intersection between antibody technology and advanced cellular immunotherapy approaches targeting CFLAR .

What methodologies are employed to study CFLAR in the tumor microenvironment?

Advanced research on CFLAR in the tumor microenvironment employs several sophisticated methodologies:

• CIBERSORTx analysis: This computational approach measures immune cell infiltration in samples stratified by CFLAR expression levels . The analysis uses a 'PERM' parameter set to 1,000 and a P-value cut-off of <0.05 to ensure statistical rigor.

• Single-cell RNA sequencing: Analysis using the 'Seurat' package identifies cell-specific CFLAR expression patterns . Quality control parameters include:

  • Selecting cells expressing between 50-9,000 genes

  • Applying a mitochondrial gene cut-off of 5%

  • Identifying 1,500 hypervariable genes

  • Adjusting 20 principal components for clustering

  • Performing UMAP dimensionality reduction

• Tumor microenvironment scoring: The 'estimate' package calculates 'StromalScore', 'ImmuneScore', and 'ESTIMATEScore' between samples with high and low CFLAR expression .

• Quantitative PCR: For validation of CFLAR expression levels using primers:

  • CFLAR forward: 5′-AGAGTGAGGCGATTTGACCTG-3′

  • CFLAR reverse: 5′-GTCCGAAACAAGGTGAGGGTT-3′

  • Normalized to GAPDH

• Multiplex immunofluorescence: Employed to simultaneously visualize CFLAR and other markers within the tumor microenvironment, allowing spatial relationship analysis .

These methodologies collectively provide a comprehensive assessment of CFLAR's role in shaping the immune landscape of tumors.

What are common issues in Western blot detection of CFLAR and how can they be resolved?

Western blot detection of CFLAR presents several challenges that can be systematically addressed:

For optimal results, researchers should validate their Western blot approach using positive control samples with known CFLAR expression patterns .

How should researchers troubleshoot flow cytometry experiments with CFLAR antibodies?

When troubleshooting flow cytometry experiments using CFLAR antibodies, consider these key factors:

• Compensation matrix issues: Single-stain controls must be run with every experiment rather than applying old compensation matrices to new samples . Using outdated matrices "could mean that the compensation matrix needs to be different, and the only way to properly create or adjust a compensation matrix is with single stain controls" .

• Beads versus cells for compensation: Be aware that "the emission spectra of a fluorophore is sometimes different if the fluorophore is on a bead vs. a cell" . When possible, use the same cell type for compensation controls as in your experimental samples.

• Autofluorescence interference: For cell types with high autofluorescence (like alveolar macrophages), consider:

  • Moving problematic markers to different fluorophore channels

  • Using autofluorescence reduction techniques

  • Implementing proper compensation and FMO controls

• Fixation effects: Optimize fixation protocols to preserve CFLAR epitopes while allowing adequate antibody access to intracellular targets.

• Gating strategy validation: Use FMO controls rather than isotype controls for more accurate gating, as "isotype controls identify problems with background staining, but don't account for spreading error from other fluorophores in the panel" .

Maintaining detailed documentation of parameter names and sample identifiers will facilitate troubleshooting and ensure reproducibility of results .

What factors affect reproducibility in CFLAR immunostaining experiments?

Several factors can impact reproducibility in CFLAR immunostaining experiments:

• Fixation and antigen retrieval: Variability in fixation times or antigen retrieval methods significantly impacts epitope accessibility . For CFLAR, "suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" .

• Antibody selection and validation: Different antibodies target distinct epitopes within CFLAR, potentially yielding different staining patterns . Validate antibodies using known positive control tissues like "mouse skeletal muscle tissue, human skeletal muscle tissue" .

• Protocol standardization: Minor variations in incubation times, temperatures, buffer compositions, or washing steps can significantly impact results. Document protocols meticulously.

• Tissue handling and processing: Variations in time to fixation, fixative composition, or paraffin embedding procedures can affect protein preservation and antibody accessibility.

• Detection systems: Different visualization methods (chromogenic vs. fluorescent) or amplification systems may yield varying sensitivity and specificity.

• Image acquisition parameters: Inconsistent exposure settings, gain adjustments, or imaging thresholds can create artificial differences between experiments.

Implementing a quality control system with standard operating procedures and regular verification using positive controls can significantly improve reproducibility .

How can researchers validate the specificity of their CFLAR antibody results?

To rigorously validate CFLAR antibody specificity, researchers should employ multiple complementary approaches:

• Genetic validation: Compare staining in wild-type samples versus those with CFLAR knockdown or knockout . This definitive approach confirms that the detected signal is truly CFLAR-specific.

• Multiple antibody validation: Use independent antibodies targeting different CFLAR epitopes to confirm consistent staining patterns . Convergent results from different antibodies provide strong evidence for specificity.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific staining should be blocked by this competition.

• Western blot correlation: Confirm that immunostaining results correlate with Western blot detection of the expected CFLAR isoforms (20-35 kDa, 50-55 kDa) .

• Positive and negative control tissues: Include tissues with known CFLAR expression patterns in each experiment . Skeletal muscle tissue from mice and humans serves as a reliable positive control .

• Recombinant protein controls: For antibodies raised against recombinant CFLAR fusion proteins, validate using the purified protein as a control .

• Cross-reactivity testing: When working with multiple species, verify specificity across species boundaries, particularly if sequence homology is not complete .

This multi-layered validation approach ensures confidence in experimental findings and facilitates meaningful interpretation of CFLAR expression data.

What are the current limitations in CFLAR antibody research methodology?

Despite significant advances, several limitations persist in CFLAR antibody research:

• Isoform specificity: Many current antibodies cannot reliably distinguish between the different CFLAR isoforms (c-FLIPL, c-FLIPR, c-FLIPS), complicating functional studies .

• Post-translational modification detection: Few antibodies specifically recognize phosphorylated, ubiquitinated, or other modified forms of CFLAR that may have distinct functions .

• Standardization challenges: Lack of standardized protocols for sample preparation, antibody dilution, and detection methods hampers cross-study comparisons .

• Tissue-specific validation: Limited validation across diverse tissue types may lead to unexpected results when applying antibodies to new experimental systems .

• Flow cytometry limitations: Technical challenges in flow cytometry include autofluorescence interference, compensation complexity, and optimal fixation/permeabilization for intracellular CFLAR detection .

• Quantification inconsistencies: Variations in image analysis approaches and quantification methods create challenges for comparing CFLAR expression levels across studies.

Addressing these limitations through development of more specific antibodies and standardized protocols will enhance the reliability and utility of CFLAR research in the future.

What emerging technologies and methods show promise for future CFLAR research?

Several innovative approaches are poised to advance CFLAR research:

• CAR-T cell therapy targeting CFLAR: Engineering of T cells with anti-CFLAR chimeric antigen receptors represents a novel therapeutic approach for cancers like colorectal cancer .

• Single-cell analysis techniques: Advanced single-cell RNA sequencing and protein detection methods provide unprecedented insights into cell type-specific CFLAR expression patterns and function .

• Spatial transcriptomics and proteomics: These technologies allow mapping of CFLAR expression within the spatial context of tissues, revealing microenvironmental influences.

• CRISPR-based functional genomics: High-throughput CRISPR screens are identifying genetic modifiers of CFLAR function and revealing new regulatory pathways .

• Advanced computational approaches: Tools like CIBERSORTx and single-sample gene set enrichment analysis enable sophisticated correlation of CFLAR expression with immune cell infiltration and function .

• Multiplex imaging technologies: These allow simultaneous visualization of CFLAR alongside multiple other proteins, providing insights into its interactions within complex signaling networks .

• Improved antibody technologies: Development of recombinant antibodies with enhanced specificity for particular CFLAR isoforms and post-translational modifications will enable more precise studies.