TNFRSF9 Antibody, FITC conjugated

What is TNFRSF9 and why is it an important research target?

TNFRSF9 (CD137/4-1BB) is a 27.9 kilodalton costimulatory receptor protein belonging to the tumor necrosis factor receptor superfamily. It plays a crucial role in T cell activation, proliferation, and survival, making it a significant target in immunotherapy research. TNFRSF9 is expressed on activated T cells, NK cells, and certain dendritic cells, serving as a key mediator in immune responses. The protein's involvement in enhancing cytotoxic T cell activity has positioned it as an important target for cancer immunotherapy development and basic immunological research .

What advantages does FITC conjugation offer for TNFRSF9 antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent detection capabilities to TNFRSF9 antibodies, eliminating the need for secondary antibody steps. This conjugation enables direct visualization in flow cytometry, immunofluorescence microscopy, and immunohistochemistry applications. The FITC fluorophore emits green fluorescence (peak emission ~520 nm) when excited by blue light (peak excitation ~495 nm), offering good sensitivity for detecting TNFRSF9 expression on cell surfaces. The direct conjugation also reduces background signal and simplifies multiplexing with other fluorophores in multicolor experiments .

What are the optimal sample preparation methods for flow cytometry when using FITC-conjugated TNFRSF9 antibodies?

For flow cytometry applications, cells should be harvested and suspended in cold PBS containing 2% FBS to maintain viability and reduce non-specific binding. A concentration of 1-5×10^6 cells per 100 μL is recommended. For best results, cells should be blocked with 5-10% normal serum from the same species as the secondary antibody (if used) for 10-15 minutes prior to staining. The FITC-conjugated TNFRSF9 antibody should be added at the manufacturer's recommended concentration (typically 1-10 μg/mL) and incubated for 30-45 minutes at 4°C in the dark. After staining, cells should be washed twice with PBS/2% FBS to remove unbound antibody before analysis. For intracellular staining, a permeabilization step using 0.1% saponin or commercial permeabilization buffers is required after fixation .

How should FITC-conjugated TNFRSF9 antibodies be stored to maintain optimal activity?

FITC-conjugated TNFRSF9 antibodies should be stored according to manufacturer recommendations, typically at 2-8°C for short-term storage (1-2 weeks) and at -20°C for long-term storage. Antibodies should be protected from light at all times due to FITC's susceptibility to photobleaching. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity. Working aliquots can be prepared and stored separately to minimize freeze-thaw cycles. Additionally, stabilizing proteins like BSA (0.1-1%) may be added to diluted antibody solutions to prevent adsorption to container surfaces and maintain antibody functionality .

What controls are essential when designing experiments with FITC-conjugated TNFRSF9 antibodies?

When designing experiments with FITC-conjugated TNFRSF9 antibodies, several controls are crucial for result validation:

• Isotype control: A FITC-conjugated antibody of the same isotype but irrelevant specificity to assess non-specific binding

• Unstained control: Cells without any antibody to establish autofluorescence baseline

• Single-color controls: When performing multicolor experiments, for compensation settings

• Positive control: Cells known to express TNFRSF9 (activated T cells or appropriate cell lines)

• Negative control: Cells known not to express TNFRSF9

• Blocking control: Pre-incubation with unconjugated anti-TNFRSF9 to confirm specificity

• FMO (Fluorescence Minus One) controls: When performing multicolor flow cytometry

These controls help distinguish true TNFRSF9 expression from technical artifacts and enable proper gating strategies for accurate data interpretation .

How can researchers optimize FITC-conjugated TNFRSF9 antibody performance in multicolor flow cytometry?

Optimizing multicolor flow cytometry with FITC-conjugated TNFRSF9 antibodies requires careful panel design and protocol refinement:

• Panel design: Choose fluorophores with minimal spectral overlap with FITC (avoid PE, which has considerable overlap). FITC works well with APC, PE-Cy7, and BV421 in multicolor panels.

• Titration: Determine the optimal antibody concentration by testing serial dilutions (typically 0.1-10 μg/mL) to identify the concentration that provides maximum signal separation with minimal background.

• Compensation setup: Use single-color controls for proper compensation setup to account for spectral overlap between fluorophores.

• Buffer optimization: Test different staining buffers; PBS with 2% FBS works well, but specialized flow cytometry buffers may reduce background further.

• Fixation consideration: If samples require fixation, use 1-2% paraformaldehyde, as higher concentrations may affect FITC fluorescence intensity.

• Instrument settings: Optimize PMT voltages for the FITC channel to position negative population appropriately while maximizing resolution.

A properly optimized protocol yields higher signal-to-noise ratios and more reliable detection of TNFRSF9-positive populations in complex samples .

What are the recommended antibody concentrations for different applications of FITC-conjugated TNFRSF9 antibodies?

These recommendations should be adjusted based on the specific antibody clone, sample type, and expression level of TNFRSF9. It is advisable to perform titration experiments for each new application or sample type to determine optimal concentration .

How can researchers validate the specificity of FITC-conjugated TNFRSF9 antibodies?

Validating antibody specificity is critical for reliable research results. For FITC-conjugated TNFRSF9 antibodies, multiple validation approaches should be employed:

• Side-by-side comparison with multiple anti-TNFRSF9 antibody clones targeting different epitopes to confirm consistent staining patterns.

• Blocking experiments using recombinant TNFRSF9 protein or unconjugated antibody of the same clone to demonstrate specific signal reduction.

• Correlation with mRNA expression through complementary techniques like RT-PCR or RNA-seq.

• Testing on TNFRSF9 knockout or knockdown models as negative controls.

• Stimulation experiments comparing resting vs. activated T cells (TNFRSF9 should increase upon activation).

• Western blot validation using the same antibody (if compatible) to confirm target molecular weight.

• Cross-validation with orthogonal methods (e.g., comparing flow cytometry results with immunohistochemistry).

Proper validation ensures experimental rigor and reproducibility, particularly in studies targeting therapeutic applications or biomarker development .

How can FITC-conjugated TNFRSF9 antibodies be utilized in studying immune checkpoint modulation?

FITC-conjugated TNFRSF9 antibodies serve as valuable tools for investigating immune checkpoint modulation in cancer immunotherapy research. Researchers can employ these antibodies to:

• Monitor TNFRSF9 expression changes on tumor-infiltrating lymphocytes (TILs) before and after immunotherapy treatments, establishing correlations between expression patterns and treatment outcomes.

• Perform co-expression analyses with other checkpoint molecules (PD-1, CTLA-4, LAG-3) through multicolor flow cytometry to characterize the immune checkpoint landscape on different T cell subsets.

• Assess TNFRSF9 upregulation in response to various stimuli, including TCR engagement, cytokines, or agonistic antibodies, providing insights into activation dynamics.

• Isolate TNFRSF9-positive T cell populations through FACS for downstream functional assays or transcriptomic analyses to identify unique characteristics of these cells.

• Develop ex vivo assays to test agonistic or antagonistic TNFRSF9-targeting therapeutics, using the FITC-conjugated antibody as a readout for receptor occupancy and modulation.

This application enables researchers to evaluate the potential of TNFRSF9 as a therapeutic target and develop biomarkers for patient stratification in clinical trials .

What technical considerations should be addressed when using FITC-conjugated TNFRSF9 antibodies for imaging tissue microenvironments?

Imaging tissue microenvironments using FITC-conjugated TNFRSF9 antibodies presents unique technical challenges that require careful consideration:

• Autofluorescence mitigation: Tissues often exhibit significant green autofluorescence that can interfere with FITC signals. Treatment with sodium borohydride (0.1% for 10 minutes) or commercial autofluorescence quenchers before antibody application can reduce this interference.

• Penetration optimization: For thick tissue sections, permeabilization protocols may need optimization. Triton X-100 (0.1-0.5%) or saponin (0.1%) can enhance antibody penetration, but excessive permeabilization may disrupt tissue architecture.

• Signal amplification strategies: For low TNFRSF9 expression, signal amplification through tyramide signal amplification (TSA) or quantum dots can enhance detection sensitivity without increasing background.

• Multiplexing techniques: When combining with other markers, sequential staining protocols or spectral unmixing algorithms may be necessary to distinguish FITC signals from other fluorophores.

• Fixation optimization: Paraformaldehyde fixation (2-4%) preserves TNFRSF9 epitopes while maintaining tissue morphology, but fixation time should be optimized (typically 15-20 minutes).

• Z-stack acquisition and deconvolution: For three-dimensional analysis, Z-stack acquisition with subsequent deconvolution can improve resolution and signal-to-noise ratio.

Addressing these considerations ensures accurate visualization and quantification of TNFRSF9 expression patterns within complex tissue microenvironments .

How can researchers investigate TNFRSF9 signaling dynamics using FITC-conjugated antibodies in combination with other techniques?

Investigating TNFRSF9 signaling dynamics requires integrating FITC-conjugated antibodies with complementary techniques to capture both spatial and temporal aspects of receptor behavior:

• Live-cell imaging: FITC-conjugated TNFRSF9 antibodies can be used for real-time visualization of receptor clustering and internalization following stimulation, though photobleaching must be minimized through reduced laser power and anti-fade reagents.

• Proximity ligation assays (PLA): Combining FITC-conjugated anti-TNFRSF9 with antibodies against potential interaction partners (TRAF1, TRAF2) in PLA reveals molecular interactions with spatial resolution.

• FRET/FLIM analysis: When paired with compatible acceptor fluorophores on antibodies targeting signaling partners, FITC-conjugated TNFRSF9 antibodies can enable FRET analysis to demonstrate molecular proximity (<10 nm) indicative of direct interaction.

• Receptor internalization studies: Flow cytometry with acid washing (pH 2.0 glycine buffer) to remove surface-bound antibodies helps differentiate between surface and internalized TNFRSF9 receptors after stimulation.

• Signaling pathway activation: Combining TNFRSF9 detection with phospho-flow cytometry targeting downstream molecules (NFκB, MAP kinases) creates a comprehensive picture of receptor engagement and signal propagation.

• Single-cell analysis platforms: Integrating FITC-conjugated TNFRSF9 staining with mass cytometry or single-cell RNA-seq links receptor expression to broader cellular phenotypes and transcriptional programs.

This multifaceted approach provides insights into how TNFRSF9 engagement translates into functional outcomes in different immune cell populations .

What considerations are important when analyzing contradictory TNFRSF9 expression data across different experimental systems?

When researchers encounter contradictory TNFRSF9 expression data across different experimental systems, several factors should be systematically evaluated:

• Antibody clone variability: Different anti-TNFRSF9 clones recognize distinct epitopes, which may be differentially accessible depending on protein conformation or microenvironment. Compare results using multiple validated clones.

• Expression kinetics: TNFRSF9 expression is highly dynamic and time-dependent after activation. Discrepancies may reflect different time points rather than true contradictions. Conduct time-course experiments spanning 6, 12, 24, 48, and 72 hours post-stimulation.

• Microenvironmental factors: TNFRSF9 expression is modulated by cytokines, cell-cell interactions, and metabolic conditions. Standardize culture conditions or thoroughly document differences between systems.

• Sample processing effects: Processing methods (enzymatic digestion, mechanical dissociation) can cleave surface proteins or induce stress responses affecting TNFRSF9 expression. Compare fresh vs. processed samples to quantify these effects.

• Species differences: Human and murine TNFRSF9 show approximately 70% homology but differ in expression patterns and regulation. Avoid direct cross-species comparisons without appropriate controls.

• Technical limitations: Flow cytometry versus microscopy versus Western blotting may yield different results due to technical parameters like sensitivity, specificity, and sample preparation requirements.

• Statistical approach: Ensure appropriate statistical methods are applied to determine if differences are statistically significant or within expected biological variation.

Researchers should systematically address these factors through carefully designed experiments with appropriate controls before concluding genuine biological differences versus methodological artifacts .

What are the most common technical issues with FITC-conjugated TNFRSF9 antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when working with FITC-conjugated TNFRSF9 antibodies. Here are the most common issues and their solutions:

• Weak signal intensity:

  • Increase antibody concentration after careful titration

  • Extend incubation time (up to overnight at 4°C)

  • Use signal amplification systems like biotinylated anti-FITC followed by streptavidin-conjugated fluorophores

  • Ensure samples are properly permeabilized for intracellular targets

  • Check for antibody degradation (avoid repeated freeze-thaw cycles)

• High background/non-specific staining:

  • Implement stricter blocking protocols (5-10% serum for 30-60 minutes)

  • Add 0.1% bovine serum albumin to staining buffer

  • Include Fc receptor blocking reagent for samples containing Fc receptor-positive cells

  • Reduce antibody concentration based on titration experiments

  • Utilize more stringent washing steps (increased number and duration)

• Photobleaching:

  • Minimize exposure to light during all protocol steps

  • Add anti-fade reagents to mounting media

  • Capture images quickly with minimal excitation intensity

  • Consider alternative fluorophores with greater photostability for critical experiments

• Spectral overlap in multicolor experiments:

  • Perform comprehensive compensation controls

  • Redesign panels to use fluorophores with less overlap with FITC

  • Apply spectral unmixing algorithms during analysis

  • Consider sequential staining and imaging for colocalization studies

• Inconsistent staining between experiments:

  • Standardize all protocol parameters (times, temperatures, antibody lots)

  • Prepare master mixes for antibody cocktails

  • Include consistent positive and negative controls in each experiment

  • Document lot-to-lot variation and adjust protocols accordingly

Addressing these issues systematically improves data reliability and experimental reproducibility .

How should researchers interpret heterogeneous TNFRSF9 expression patterns detected with FITC-conjugated antibodies?

Interpreting heterogeneous TNFRSF9 expression patterns requires careful analysis and consideration of multiple factors:

• Biological relevance assessment:

  • Determine if heterogeneity correlates with known cell subsets by co-staining with lineage markers

  • Investigate whether TNFRSF9 expression levels correlate with functional differences through sorting and functional assays

  • Compare expression patterns across healthy and disease samples to identify disease-specific alterations

• Quantitative analysis approaches:

  • Apply appropriate gating strategies that account for population heterogeneity

  • Use bimodality indices or mixture modeling to objectively define positive and negative populations

  • Implement visualization techniques like t-SNE or UMAP to reveal complex expression patterns within multidimensional data

• Resolution of technical versus biological heterogeneity:

  • Validate observations using alternative antibody clones or detection methods

  • Test single-cell clones to determine if heterogeneity is maintained in clonal populations

  • Compare fresh versus cultured samples to identify culture-induced artifacts

• Temporal dynamics consideration:

  • Perform time-course experiments to determine if heterogeneity reflects different activation states

  • Assess if cell cycle phase correlates with expression level through co-staining with cell cycle markers

• Microenvironmental context:

  • For tissue samples, correlate expression with spatial location and neighboring cell types

  • Evaluate the impact of local cytokine milieu on expression patterns

What methodological modifications are needed when staining different sample types with FITC-conjugated TNFRSF9 antibodies?

This adaptable approach ensures optimal staining across diverse sample types while maintaining specificity and signal quality. Each protocol should be validated with appropriate positive and negative controls before experimental application .

How can researchers accurately quantify TNFRSF9 expression levels using FITC-conjugated antibodies?

Accurate quantification of TNFRSF9 expression levels requires methodological rigor and standardization:

• Antibody binding capacity (ABC) determination:

  • Use quantitative fluorescence calibration beads with known FITC molecules per bead

  • Generate calibration curves relating mean fluorescence intensity (MFI) to molecules of equivalent soluble fluorochrome (MESF)

  • Calculate the number of antibodies bound per cell by comparing sample MFI to the calibration curve

  • Account for the fluorophore-to-protein ratio of the specific antibody lot

• Reference standards implementation:

  • Include consistent biological reference samples (e.g., stimulated PBMCs) in each experiment

  • Normalize experimental values to reference standards to account for day-to-day variations

  • Use stabilized cell preparations as long-term reference standards when available

• Instrument standardization:

  • Implement regular quality control using fluorescent beads

  • Record PMT voltages and instrument settings for reproducibility

  • Consider using shared resource instruments with rigorous QC programs for longitudinal studies

• Signal linearity validation:

  • Perform antibody titration to ensure measurements are made in the linear range of detection

  • Create dilution series of high-expressing samples to confirm signal proportionality

  • Evaluate potential signal saturation at high expression levels

• Data normalization strategies:

  • Calculate relative expression using ratio to isotype control

  • Employ staining index calculations: (MFI positive - MFI negative)/(2 × SD of negative)

  • Consider using the robust coefficient of variation (rCV) for heterogeneous populations

• Advanced flow cytometric approaches:

  • Implement fluorescence minus one (FMO) controls for accurate gating

  • Consider density-based clustering algorithms for heterogeneous populations

  • Apply standardized gating strategies across experiments

These approaches enable reliable quantitative comparisons of TNFRSF9 expression across different experimental conditions, time points, and laboratories .

How are FITC-conjugated TNFRSF9 antibodies being utilized in conjunction with single-cell technologies?

FITC-conjugated TNFRSF9 antibodies have become valuable components in cutting-edge single-cell analysis platforms, enabling researchers to correlate TNFRSF9 expression with broader cellular phenotypes:

• Single-cell RNA sequencing integration:

  • FITC-conjugated TNFRSF9 antibodies are being incorporated into CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) panels to simultaneously capture surface protein expression and transcriptome information from individual cells

  • This approach reveals correlations between TNFRSF9 protein expression and transcriptional programs, identifying gene signatures associated with different TNFRSF9 expression levels

  • Researchers can map TNFRSF9-expressing cells onto single-cell transcriptomic landscapes to understand their developmental trajectories and functional states

• Mass cytometry applications:

  • Though not directly using FITC, the same anti-TNFRSF9 clones available as FITC conjugates are being adapted for mass cytometry (CyTOF) by metal isotope conjugation

  • This enables simultaneous measurement of TNFRSF9 along with 40+ other markers to comprehensively characterize immune cell subsets

  • The high-dimensional data generated allows identification of novel TNFRSF9-expressing cell populations with unique combinatorial marker profiles

• Spatial transcriptomics:

  • FITC-conjugated TNFRSF9 antibodies are being combined with spatial transcriptomics technologies to map expression patterns within tissue microenvironments

  • This approach preserves spatial context, enabling researchers to understand how TNFRSF9-expressing cells interact with other immune and stromal components

  • The integration of spatial and molecular information provides insights into the functional organization of immune responses

• Single-cell functional assays:

  • FACS-sorted TNFRSF9-positive and negative populations (using FITC-conjugated antibodies) undergo single-cell functional assays to correlate expression with cytokine production, cytotoxicity, or proliferative capacity

  • Microfluidic platforms enable real-time tracking of individual TNFRSF9-expressing cells to assess functional heterogeneity within seemingly uniform populations

These integrated approaches are revealing previously unappreciated heterogeneity within TNFRSF9-expressing populations and providing insights into their functional roles in immune responses .

What are the latest advancements in using FITC-conjugated TNFRSF9 antibodies for monitoring immunotherapy responses?

Recent advances have expanded the utility of FITC-conjugated TNFRSF9 antibodies in monitoring immunotherapy responses across multiple domains:

• Predictive biomarker development:

  • Researchers are using FITC-conjugated TNFRSF9 antibodies to establish baseline TNFRSF9 expression profiles on tumor-infiltrating lymphocytes (TILs) prior to immunotherapy

  • Emerging data suggests that TNFRSF9 expression patterns on specific T cell subsets may predict response to immune checkpoint inhibitors

  • Standardized flow cytometry panels incorporating TNFRSF9 alongside established markers (PD-1, CTLA-4) are being developed for clinical implementation

• Pharmacodynamic monitoring:

  • Serial blood and tumor sampling with FITC-conjugated TNFRSF9 antibody staining enables tracking of therapy-induced changes in TNFRSF9 expression

  • This approach reveals the kinetics of T cell activation following various immunotherapeutic interventions

  • Researchers are developing methodologies to correlate TNFRSF9 expression changes with clinical outcomes to establish relevant pharmacodynamic thresholds

• Receptor occupancy assessment:

  • For clinical trials of TNFRSF9-targeting agents, FITC-conjugated antibodies that bind non-overlapping epitopes are being used to quantify free versus occupied receptors

  • This enables precise dose optimization and helps establish target engagement metrics

  • Differential epitope binding assays using multiple fluorophore-conjugated antibodies allow more sophisticated receptor occupancy analyses

• Resistance mechanism identification:

  • Analysis of TNFRSF9 expression on TILs from treatment-resistant versus responsive patients is revealing potential resistance mechanisms

  • Integration with exhaustion marker panels helps distinguish between different types of T cell dysfunction

  • Correlation with tumor microenvironment features provides context for interpreting TNFRSF9 expression data

• Liquid biopsy applications:

  • FITC-conjugated TNFRSF9 antibodies are being applied to circulating immune cell populations as non-invasive biomarkers

  • This approach allows more frequent monitoring compared to tumor biopsies

  • Research is ongoing to determine the concordance between TNFRSF9 expression on circulating versus tumor-infiltrating T cells

These advances are enhancing the precision of immunotherapy response monitoring and contributing to more personalized treatment approaches .

How can researchers effectively validate experimental findings across species using FITC-conjugated TNFRSF9 antibodies?

Cross-species validation of TNFRSF9 biology presents unique challenges that researchers can address through careful experimental design:

Thoughtful implementation of these strategies enables more reliable translation between preclinical models and human studies .

What methodological innovations are emerging for multiplexed detection systems incorporating FITC-conjugated TNFRSF9 antibodies?

Emerging methodological innovations are expanding the capabilities of multiplexed detection systems that incorporate FITC-conjugated TNFRSF9 antibodies:

• Spectral flow cytometry advancements:

  • Full-spectrum analysis overcomes traditional limitations of FITC in multicolor panels by utilizing the entire emission spectrum rather than filtered bandpass detection

  • This approach enables inclusion of FITC-conjugated TNFRSF9 antibodies in panels with 30+ parameters

  • Advanced unmixing algorithms separate the FITC signal from spectrally similar fluorophores with unprecedented precision

  • Researchers can now include multiple green-emitting fluorophores (including FITC) in a single panel

• Multiplex immunohistochemistry platforms:

  • Cyclic immunofluorescence methods allow sequential staining, imaging, and signal removal

  • This enables combination of FITC-conjugated TNFRSF9 antibodies with dozens of additional markers on a single tissue section

  • Multispectral imaging systems with advanced unmixing capabilities extract clean FITC signals even in autofluorescent tissues

  • Computational registration of multiple imaging rounds creates comprehensive spatial maps of TNFRSF9 expression in complex microenvironments

• Proximity-based detection systems:

  • Techniques like proximity ligation assay (PLA) and proximity extension assay (PEA) are being adapted to work with FITC-conjugated antibodies

  • These approaches enable detection of TNFRSF9 protein interactions with unprecedented specificity

  • Multiplexed PLA systems can simultaneously visualize multiple interaction partners using spectral coding

• Barcoding strategies:

  • Cell barcoding approaches allow samples from different conditions to be combined, stained with FITC-conjugated TNFRSF9 antibodies, and analyzed together

  • This minimizes technical variation and enables higher-throughput screening

  • Computational debarcoding accurately reassigns cells to their original samples while maintaining data quality

• Artificial intelligence integration:

  • Machine learning algorithms are being developed to automatically identify and characterize TNFRSF9-expressing cells in complex datasets

  • These approaches standardize analysis across laboratories and reduce operator-dependent variation

  • Deep learning models incorporate spatial context to identify subtle patterns in TNFRSF9 expression not apparent through conventional analysis

These innovations are expanding the contexts in which FITC-conjugated TNFRSF9 antibodies can be applied while enhancing data quality and throughput .