TNFRSF25 Antibody, FITC conjugated

What is TNFRSF25 and why is it an important research target?

TNFRSF25 (TNF receptor superfamily member 25) is a cell surface receptor protein with a canonical structure of 417 amino acid residues and a molecular weight of approximately 45.4 kDa. It serves as a receptor for TNFSF12/APO3L/TWEAK and is highly expressed in thymocytes and lymphocytes. This protein has gained significant research interest because it functions as a marker for Regulatory T Cells and plays roles in immune regulation and inflammatory processes. Up to 12 different isoforms have been reported for this protein, making it a complex but valuable target for immunological studies . The protein undergoes post-translational modifications, including glycosylation, which can affect its function and detection capabilities in experimental settings.

What are the optimal sample preparation methods for TNFRSF25 antibody, FITC conjugated detection in flow cytometry?

For optimal detection of TNFRSF25 using FITC-conjugated antibodies in flow cytometry, sample preparation should follow these methodological steps:

• Harvest cells (typically lymphocytes or thymocytes) and wash twice in cold PBS containing 1% BSA.

• Adjust cell concentration to 1×10^6 cells/100 μl in staining buffer.

• For surface staining (recommended for TNFRSF25), block Fc receptors using appropriate blocking reagent for 15 minutes at 4°C.

• Without washing, add optimized concentration of FITC-conjugated TNFRSF25 antibody (typically 0.25-1 μg per 10^6 cells).

• Incubate for 30 minutes at 4°C in the dark to prevent photobleaching of FITC.

• Wash cells twice with staining buffer to remove unbound antibody.

• Resuspend in appropriate buffer containing viability dye if needed.

• Analyze promptly, or fix with 2% paraformaldehyde if analysis must be delayed.

This protocol maximizes signal-to-noise ratio while preserving cellular integrity and antigen accessibility. Researchers should include appropriate isotype-matched FITC-conjugated control antibodies to accurately set compensation and determine background fluorescence levels.

How should titration experiments be designed to determine optimal concentration of TNFRSF25 antibody, FITC conjugated?

Titration experiments for FITC-conjugated TNFRSF25 antibodies should follow a systematic approach to determine the optimal signal-to-noise ratio. Begin with a serial dilution series spanning at least 4-5 concentrations (typically ranging from 0.1-5 μg/mL). Use consistent cell numbers (1×10^6 cells per sample) and experimental conditions across all samples. After staining, calculate the Stain Index for each concentration using the formula:

SI = (MFI positive - MFI negative) / (2 × SD of MFI negative)

Where MFI represents Mean Fluorescence Intensity and SD is Standard Deviation.

Plot the Stain Index against antibody concentration to identify the optimal concentration, which typically appears at the plateau of the curve before signal saturation. This methodical approach ensures efficient antibody usage while maximizing detection sensitivity and specificity for TNFRSF25 expression analysis. Include both positive controls (cells known to express TNFRSF25, such as activated T cells) and negative controls to validate your titration results.

What strategies can overcome autofluorescence issues when using FITC-conjugated TNFRSF25 antibodies in tissues with high background?

When working with tissues that exhibit high autofluorescence in the FITC channel (such as liver, kidney, or brain tissues), several methodological approaches can maximize signal-to-noise ratio:

• Implement tissue-specific autofluorescence quenching: Treat tissue sections with 0.1-0.3% Sudan Black B in 70% ethanol for 20 minutes at room temperature before antibody incubation.

• Use spectral unmixing algorithms during image acquisition and analysis to computationally separate FITC signal from autofluorescence.

• Consider time-gated detection methods that exploit the typically longer fluorescence lifetime of FITC compared to endogenous fluorophores.

• Compare results with alternative conjugates that emit at longer wavelengths (such as PE or Cy5-conjugated TNFRSF25 antibodies) which often face less interference from tissue autofluorescence.

• Implement additional blocking steps with 5-10% normal serum matching the species of your samples for 1-2 hours before antibody incubation.

For particularly challenging samples, combining multiple approaches may be necessary. Always include unstained control sections to accurately assess autofluorescence levels and properly calculate true positive signal from background.

How do different permeabilization methods affect intracellular detection of TNFRSF25 using FITC-conjugated antibodies?

Different permeabilization methods significantly impact the detection efficiency of intracellular TNFRSF25 using FITC-conjugated antibodies. This comparative analysis highlights the most effective approaches:

For optimal intracellular detection of TNFRSF25, a sequential approach is recommended: first fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with 0.1% saponin in PBS containing 0.5% BSA. This method provides the best balance between epitope preservation and antibody accessibility while maintaining FITC fluorescence intensity. For particularly challenging samples, extending permeabilization time rather than increasing detergent concentration generally yields better results while preserving both cellular integrity and FITC signal.

How can TNFRSF25 antibody, FITC conjugated be effectively used in multiparameter flow cytometry panels?

Designing effective multiparameter flow cytometry panels incorporating FITC-conjugated TNFRSF25 antibodies requires strategic consideration of spectral overlap and marker co-expression. Since FITC has potential spillover into PE channels, implement these methodological approaches:

• Strategic panel design: Position the FITC-conjugated TNFRSF25 antibody in a channel separate from markers with similar expression patterns or biological relationships.

• Optimal fluorophore selection: Pair FITC with fluorophores having minimal spectral overlap, such as APC, APC-Cy7, and BV786.

• Comprehensive compensation controls: Prepare single-stained controls for each fluorophore in your panel using the same cells or compensation beads with matching antibody concentrations.

• Titration within the full panel: Re-titrate the FITC-conjugated TNFRSF25 antibody within the complete staining panel, as optimal concentration may differ from single-stain experiments.

• Consider the biological context: When studying Regulatory T cells, pair TNFRSF25-FITC with markers like CD4-APC, CD25-PE-Cy7, and FOXP3-BV421 for comprehensive phenotyping.

This systematic approach ensures accurate identification of TNFRSF25-expressing cell populations within complex samples while minimizing data artifacts from improper compensation or suboptimal staining parameters.

What are the methodological differences when using TNFRSF25 antibody, FITC conjugated for in vivo imaging versus ex vivo analysis?

In vivo imaging with FITC-conjugated TNFRSF25 antibodies presents distinct methodological challenges compared to ex vivo applications. This comparative analysis outlines key differences and optimization strategies:

For in vivo applications:

• Dosage optimization: Typically requires 1-5 μg/g body weight compared to 0.1-1 μg/10^6 cells for ex vivo staining.

• Administration route: Intravenous injection provides best systemic distribution but limits tissue penetration.

• Timing considerations: Peak signal-to-background ratio occurs 4-6 hours post-injection before significant clearance.

• Photobleaching risk: Minimize exposure time and intensity during intravital microscopy to preserve signal.

• Autofluorescence management: Implement image processing algorithms to distinguish specific signal from tissue autofluorescence.

For ex vivo applications after in vivo administration:

• Tissue processing: Rapid processing in cold buffers preserves FITC signal integrity.

• Signal amplification: Secondary anti-FITC antibodies conjugated to brighter fluorophores can recover partially degraded signals.

• Fixation considerations: Use 2% paraformaldehyde to minimize FITC quenching while maintaining tissue architecture.

The primary limitations of in vivo FITC imaging include relatively shallow tissue penetration (200-500 μm) and potential signal attenuation in highly vascularized organs. For deep tissue imaging, consider alternative near-infrared conjugates with better tissue penetration properties or implement multiphoton microscopy techniques to enhance FITC detection in deeper tissue layers.

How can dual staining with TNFRSF25 antibody, FITC conjugated and its ligand be optimized for receptor-ligand interaction studies?

Optimizing dual staining protocols for TNFRSF25 and its ligand (TNFSF12/APO3L/TWEAK) requires careful consideration of binding interference and detection strategies. This methodological approach maximizes detection efficiency while preserving biological interactions:

• Sequential staining approach: First label TNFSF12/APO3L/TWEAK with a spectrally distinct fluorophore (e.g., PE or APC) using direct conjugation or primary-secondary antibody combinations.

• Partial fixation step: Apply a mild fixation (0.5-1% paraformaldehyde for 5-10 minutes) to stabilize the ligand-receptor complex without denaturing epitopes.

• TNFRSF25 detection: Apply the FITC-conjugated TNFRSF25 antibody, ensuring it recognizes an epitope distinct from the ligand-binding domain to prevent competitive interference.

• Controls optimization: Include single-stained samples, competitive binding controls (pre-incubation with unlabeled antibodies), and biological controls (cells known to lack either receptor or ligand expression).

• Image analysis strategy: Implement colocalization analysis using Pearson's or Mander's coefficients to quantify receptor-ligand interactions, with values >0.7 typically indicating significant interaction.

This approach enables quantitative assessment of TNFRSF25-ligand interactions while minimizing artifacts from antibody cross-reactivity or competitive binding. For dynamic interaction studies, consider pulse-chase experimental designs with temporally separated staining steps to capture interaction kinetics.

How can researchers validate the specificity of TNFRSF25 antibody, FITC conjugated against potential cross-reactivity with other TNFRSF family members?

Validating FITC-conjugated TNFRSF25 antibody specificity requires a multi-faceted approach to eliminate potential cross-reactivity with structurally similar TNFRSF family members. Implement these methodological steps:

• Competitive inhibition assays: Pre-incubate the antibody with recombinant TNFRSF25 protein (5-10 μg/mL) before cell staining. A specific antibody will show significant signal reduction compared to non-blocked controls.

• Knockdown/knockout validation: Test the antibody on TNFRSF25 knockdown or knockout cell lines generated using siRNA or CRISPR-Cas9. Specific antibodies will show proportional signal reduction matching the degree of target reduction.

• Cross-adsorption testing: Evaluate antibody binding to a panel of recombinant proteins representing related TNFRSF family members (particularly TNFRSF12 due to nomenclature overlap) using ELISA or protein microarrays.

• Multi-species reactivity assessment: Test the antibody against TNFRSF25 orthologs from mouse, rat, or other species mentioned in the product specifications to confirm expected cross-reactivity patterns .

• Epitope mapping: Determine the specific recognition site using truncated protein constructs or peptide arrays to ensure the epitope is unique to TNFRSF25.

What are the most effective methods to troubleshoot weak or absent signals when using TNFRSF25 antibody, FITC conjugated?

When encountering weak or absent signals with FITC-conjugated TNFRSF25 antibodies, implement this systematic troubleshooting workflow to identify and resolve technical issues:

• Antibody functionality assessment:

  • Check fluorophore integrity by measuring antibody absorbance at 495nm (FITC absorption peak)

  • Verify performance with positive control samples (activated T cells or thymocytes)

  • Test against recombinant TNFRSF25 protein in a direct ELISA format

• Sample-related optimizations:

  • Increase cell number (2-5×10^6 cells) to improve rare population detection

  • Modify fixation protocol (reduce paraformaldehyde concentration to 2% or shorten fixation time)

  • Enhance epitope retrieval for tissue sections (try citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Staining protocol refinements:

  • Extend incubation time (45-60 minutes) at optimal temperature (typically 4°C for surface staining)

  • Reduce washing steps to prevent cell loss

  • Add protein transport inhibitors (like Brefeldin A or Monensin) before staining if detecting induced TNFRSF25 expression

• Instrument optimization:

  • Increase PMT voltage for the FITC channel

  • Verify laser alignment and optical filter integrity

  • Adjust compensation settings to prevent signal underestimation

Document all troubleshooting steps in a systematic matrix, changing only one variable at a time to identify the most critical parameters affecting detection sensitivity. This methodical approach pinpoints specific issues while establishing optimal conditions for future experiments with FITC-conjugated TNFRSF25 antibodies.

How should researchers properly store and handle TNFRSF25 antibody, FITC conjugated to maintain optimal performance over time?

Proper storage and handling of FITC-conjugated TNFRSF25 antibodies is critical for maintaining long-term performance. Follow these evidence-based practices to preserve antibody functionality:

Short-term storage (1-2 weeks):

• Store at 4°C protected from light in amber vials or wrapped in aluminum foil

• Add sodium azide (0.02-0.05%) as a preservative if not already present

• Avoid repeated freeze-thaw cycles which accelerate FITC degradation

Long-term storage (>2 weeks):

• Aliquot into single-use volumes (typically 10-25 μL) before freezing at -20°C

• For extended storage (>6 months), maintain at -80°C

• Document preparation date and freeze-thaw cycles for each aliquot

Handling during experiments:

• Thaw aliquots rapidly at room temperature while protected from light

• Centrifuge briefly (10,000g for 30 seconds) before opening to collect reagent at the bottom

• Use non-binding, low-retention polypropylene tubes to prevent antibody adsorption

• Keep on ice and protected from light during experimental procedures

Performance monitoring:

• Periodically test signal intensity against reference standards or control samples

• Monitor changes in signal-to-noise ratio over time as an indicator of conjugate degradation

• Consider implementing a quality control system with regular validation of antibody performance

By following these methodological approaches, researchers can significantly extend the functional lifespan of FITC-conjugated TNFRSF25 antibodies, ensuring consistent experimental results while maximizing reagent value.

What are the most appropriate gating strategies for analyzing TNFRSF25 expression using FITC-conjugated antibodies in various immune cell subsets?

Developing optimal gating strategies for TNFRSF25-FITC analysis requires a hierarchical approach that accommodates different immune cell subsets and expression patterns. This comprehensive methodology ensures accurate identification of positive populations:

• Initial preprocessing steps:

  • Apply time gate to exclude flow rate anomalies

  • Use FSC-A vs. FSC-H to exclude doublets

  • Gate viable cells using appropriate viability dye

• Subset-specific gating approaches:

For T cell analysis:

• Gate CD3+ T cells → CD4+ and CD8+ subsets → Apply TNFRSF25 positivity gates on each subset

• For regulatory T cells: CD3+ → CD4+ → CD25high → FOXP3+ → Analyze TNFRSF25 expression

For B cell analysis:

• Gate CD19+ B cells → Further subset by CD27 and IgD → Apply TNFRSF25 gates

• Correlate with activation markers (CD86, CD69) to assess relationship with activation state

• Defining TNFRSF25 positivity:

  • Use fluorescence-minus-one (FMO) controls rather than isotype controls for precise boundary setting

  • Consider bimodal expression patterns by using density plots rather than simple histograms

  • When appropriate, define positive populations using standard deviation from negative population means

• Advanced analysis considerations:

  • Implement probability contour plots with 5% contour intervals for better visualization of rare TNFRSF25+ events

  • Apply dimensionality reduction techniques (tSNE, UMAP) for unbiased identification of TNFRSF25+ subpopulations

This systematic approach enables accurate quantification of TNFRSF25 expression across different immune compartments while minimizing artifacts from improper gating or biological variability between samples.

How do expression levels of TNFRSF25 correlate with functional outcomes in different experimental models?

TNFRSF25 expression levels exhibit distinct correlations with functional outcomes across various experimental models, providing valuable insights for data interpretation:

In T cell activation models:
High TNFRSF25 expression (MFI >1000) correlates with increased susceptibility to activation-induced cell death (AICD) through enhanced caspase-8 activation and mitochondrial membrane permeabilization. Cells with intermediate expression levels (MFI 400-1000) demonstrate heightened proliferative responses to TCR stimulation with increased IL-2 production, while maintaining resistance to apoptosis.

In regulatory T cell (Treg) function:
TNFRSF25 expression positively correlates with suppressive capacity, with TNFRSF25high Tregs exhibiting 2-3 fold greater suppressive activity compared to TNFRSF25low Tregs in standard suppression assays. This functional difference is associated with increased FOXP3 stability and enhanced production of immunoregulatory cytokines (IL-10, TGF-β).

In inflammatory disease models:
Temporal analysis of TNFRSF25 expression during disease progression reveals biphasic patterns: initial upregulation during acute inflammation followed by downregulation during resolution phases. This expression pattern serves as a potential biomarker for disease activity and treatment response assessment.

These correlation patterns provide critical context for interpreting TNFRSF25 expression data, moving beyond descriptive phenotyping to functional relevance in immunological processes. Researchers should consider these functional correlations when designing experiments and analyzing TNFRSF25 expression in their specific experimental systems.

What statistical approaches are most appropriate for analyzing TNFRSF25 expression data from flow cytometry experiments?

Selecting appropriate statistical methods for analyzing TNFRSF25 flow cytometry data depends on the experimental design and data characteristics. This methodological framework outlines the most suitable approaches:

For comparing TNFRSF25 expression between two defined groups:

• Parametric approach: Independent t-test when data follows normal distribution

• Non-parametric alternative: Mann-Whitney U test when normality assumptions are violated

• Effect size calculation: Cohen's d to quantify the magnitude of difference between groups

For multiple group comparisons:

• One-way ANOVA with post-hoc Tukey's test for normally distributed data

• Kruskal-Wallis with Dunn's post-hoc test for non-parametric analysis

• Control for multiple comparisons using Bonferroni or FDR correction methods

For correlation analysis with continuous variables:

• Pearson correlation coefficient for linear relationships with normally distributed data

• Spearman's rank correlation for non-parametric or non-linear relationships

• Consider multivariate regression models to account for confounding variables

For analysis of bimodal or complex expression patterns:

• Finite mixture modeling to identify and characterize subpopulations

• Probability binning or Kolmogorov-Smirnov statistics to detect subtle distribution shifts

Sample size considerations:

• Power analysis based on preliminary data to determine adequate sample sizes

• Typically requires 15-20 samples per group to detect a 30% difference in expression with 80% power

This framework ensures robust statistical analysis of TNFRSF25 expression data while avoiding common pitfalls such as inappropriate test selection or inadequate handling of non-normal distributions often encountered in flow cytometry data.

How does FITC-conjugated TNFRSF25 antibody performance compare with other fluorophore conjugates for different applications?

FITC-conjugated TNFRSF25 antibodies demonstrate distinct performance characteristics compared to other fluorophore conjugates across various applications. This comparative analysis guides optimal conjugate selection:

For flow cytometry applications, FITC-conjugated TNFRSF25 antibodies provide adequate sensitivity for detecting moderate to high expression levels but may struggle with rare or low-expressing populations compared to PE conjugates, which offer approximately 5-6 fold higher sensitivity. For imaging applications, the quick photobleaching of FITC (typical half-life of 10-20 seconds under continuous illumination) makes Alexa Fluor 488 conjugates preferable for extended imaging sessions despite higher costs.

What innovative approaches combine TNFRSF25 detection with functional assays to provide deeper insights into cellular responses?

Innovative methodological approaches that integrate TNFRSF25-FITC detection with functional assays provide multidimensional insights into immune cell biology. These advanced techniques enable correlation between receptor expression and functional outcomes:

• Flow cytometry-based phosphoprotein analysis: Combine TNFRSF25-FITC surface staining with intracellular phospho-flow detection of downstream signaling molecules (p-ERK, p-NF-κB p65) following receptor ligation. This approach reveals the relationship between receptor density and signaling capacity at the single-cell level.

• TNFRSF25-targeted functional sorting: Use FITC-conjugated TNFRSF25 antibodies to sort cells into TNFRSF25high and TNFRSF25low populations followed by:

  • Cytokine secretion analysis using multiplex bead arrays

  • Proliferation assessment with cell division tracking dyes

  • Transcriptional profiling with RNA-seq or NanoString technology

• Live-cell imaging with real-time functional readouts: Combine TNFRSF25-FITC detection with:

  • Calcium flux indicators (Fluo-4, Indo-1) to correlate receptor expression with calcium signaling

  • Mitochondrial membrane potential dyes to assess apoptotic sensitivity

  • Nuclear translocation reporters for transcription factors activated downstream of TNFRSF25

• Correlative microscopy approaches: Implement correlative light and electron microscopy (CLEM) to examine TNFRSF25 distribution in relation to ultrastructural features like mitochondrial morphology or immune synapses.

• Receptor internalization and trafficking analysis: Use TNFRSF25-FITC antibodies in pulse-chase experiments combined with endosomal markers to track receptor fate after ligand binding, correlating trafficking patterns with functional outcomes.

These integrated approaches move beyond descriptive phenotyping to establish causal relationships between TNFRSF25 expression patterns and cellular function, providing mechanistic insights into receptor biology in both physiological and pathological contexts.

How can researchers effectively integrate TNFRSF25-FITC antibody data with genomic and proteomic datasets for systems biology approaches?

Integrating TNFRSF25-FITC antibody data with genomic and proteomic datasets requires systematic methodological approaches to enable comprehensive systems biology analysis:

• Multi-omics data generation and preprocessing:

  • Sort TNFRSF25high and TNFRSF25low populations using FITC-conjugated antibodies

  • Process sorted populations for RNA-seq, ATAC-seq, and proteomics analysis

  • Implement batch correction and data normalization to enable cross-platform comparison

• Data integration frameworks:

  • Apply canonical correlation analysis (CCA) to identify relationships between TNFRSF25 expression and transcriptional/proteomic signatures

  • Implement MOFA (Multi-Omics Factor Analysis) to extract factors explaining variance across datasets

  • Use network-based approaches like WGCNA (Weighted Gene Co-expression Network Analysis) to identify modules associated with TNFRSF25 expression levels

• Pathway enrichment and functional annotation:

  • Perform integrated pathway analysis using tools like Ingenuity Pathway Analysis or Metascape

  • Identify transcription factor networks associated with different TNFRSF25 expression states

  • Map protein-protein interaction networks centered on TNFRSF25 and its signaling partners

• Validation and hypothesis testing:

  • Confirm key relationships using orthogonal methods (qPCR, Western blot)

  • Apply CRISPR-based perturbation of identified nodes to validate network predictions

  • Develop predictive models of cellular responses based on TNFRSF25 expression patterns

• Data visualization and sharing:

  • Create interactive visualizations of integrated datasets using tools like Cytoscape or ggplot2

  • Deposit datasets in appropriate repositories (GEO, PRIDE) with standardized metadata

  • Provide computational workflows for reproducibility

This systematic approach transforms descriptive TNFRSF25 expression data into mechanistic insights about its role within cellular networks. The integration of flow cytometry data with genomic and proteomic information reveals emergent properties not apparent from individual datasets, enabling holistic understanding of TNFRSF25 biology in complex systems.