Tf Antibody, FITC conjugated

What is Tissue Factor (TF) and why is it a significant research target?

Tissue Factor (TF), also known as CD142, Coagulation Factor III, F3, or Thromboplastin, is a transmembrane glycoprotein that initiates the extrinsic pathway of blood coagulation. Beyond its role in coagulation, TF is aberrantly expressed in various solid cancers and is implicated in tumor progression through both its procoagulant activity and its capacity to induce intracellular signaling when complexed with Factor VIIa (FVIIa) . TF is frequently expressed in highly invasive triple negative breast cancer (TNBC) and pancreatic adenocarcinoma (PaC), which are among the most challenging tumor types with poor survival rates and limited therapeutic options . The pathological presence of TF in cancer cells has been linked to tumor-initiated thrombosis and cancer metastasis, making it a promising target for therapeutic interventions .

What distinguishes TF Antibody, FITC conjugated from other research antibodies?

TF Antibody, FITC conjugated is a purified antibody validated for specificity and sensitivity, with the fluorescent dye FITC chemically attached to enable direct visualization in fluorescence-based applications . The FITC molecule has excitation and emission spectrum peak wavelengths of approximately 495 nm and 519 nm, producing green fluorescence when excited with the appropriate wavelength light . Unlike other research antibodies that may require secondary detection systems, FITC-conjugated antibodies allow for direct detection, simplifying experimental workflows and reducing background in multi-color experiments. The specific TF antibody products are validated across multiple applications including immunofluorescence (IF), immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry (FACS) .

How does the FITC conjugation process affect antibody functionality?

The conjugation of FITC to antibodies must be carefully controlled to maintain antibody function. Traditional random conjugation methods can potentially impact the antigen-binding domain's functionality. Advanced site-specific conjugation technologies (similar to FluoSite™ mentioned for other antibodies) ensure that the labeling site is positioned away from the antigen-binding domain, preserving antibody functionality . When evaluating a TF antibody with FITC conjugation, researchers should consider the fluorophore-to-protein ratio (F/P ratio) as this impacts both signal strength and potential interference with antibody binding. Optimally conjugated antibodies maintain high affinity binding while providing sufficient fluorescence for detection .

What are the optimal protocols for using TF Antibody, FITC conjugated in flow cytometry?

When using TF Antibody, FITC conjugated for flow cytometry, researchers should:

• Begin with cell concentrations of 1 × 10^6 cells/ml in appropriate buffer (typically PBS with 1-2% BSA)

• Use titrated antibody concentrations to determine optimal signal-to-noise ratio (typically starting with manufacturer recommendations of approximately 10 μg/ml)

• Incubate cells with antibody for 30-45 minutes at 4°C protected from light

• Wash cells twice with buffer to remove unbound antibody

• Analyze immediately or fix with 1-2% paraformaldehyde if analysis must be delayed

• Include appropriate controls:

  • Unstained cells

  • Isotype control antibody-FITC to assess non-specific binding

  • Positive control samples with known TF expression

Flow cytometric analysis has been used successfully to assess the binding activity of anti-TF antibodies to cancer cell lines with high TF expression, such as the gastric cancer line 44As3 and pancreatic cancer line BxPC3 . The method allows quantitative assessment of binding affinity differences between various anti-TF constructs.

How can researchers effectively validate the specificity of TF Antibody, FITC conjugated?

To validate TF antibody specificity, researchers should implement a multi-faceted approach:

• Positive and negative cell line panel testing: Use cell lines with documented high TF expression (e.g., BxPC3 pancreatic cancer cells, 44As3 gastric cancer cells) and negative control cell lines

• Competitive binding assays: Pre-incubate with unconjugated TF antibody or recombinant TF protein before adding the FITC-conjugated antibody

• siRNA or CRISPR knockdown: Create TF knockdown cell lines and demonstrate reduced binding

• Western blot correlation: Confirm that fluorescence intensity correlates with protein expression levels detected by Western blot

• Tissue validation: Compare staining patterns with literature-reported TF expression in tissue sections

In published research, TF antibody specificity has been validated using tissue factor-transfected HEK293F or A431 cells and by comparison with bead-coupled TF-ECDHis using Fluorimetric Microvolume Assay Technology .

What storage and handling practices ensure optimal performance of TF Antibody, FITC conjugated?

For maximum stability and performance:

• Store the antibody at 2-8°C and avoid freezing, as noted in product specifications

• Protect from extended light exposure to prevent photobleaching of the FITC fluorophore

• If lyophilized, reconstitute in sterile water or buffer specified by the manufacturer

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

• For long-term storage of reconstituted antibody, aliquot and store at -20°C

• When working with the antibody, minimize exposure to light and maintain cold temperatures

• Check the expiration date and lot-specific quality control data provided by the manufacturer

• Consider adding sodium azide (0.02%) to prevent microbial growth if preparing working solutions, but be aware this may interfere with some applications (especially those involving peroxidase)

Lyophilized TF Antibody, FITC conjugated is typically presented in a stabilizing buffer containing PBS pH 7.4, 20 mg/ml BSA, 0.02% Sodium Azide, and 4% Trehalose to maintain stability .

How can TF Antibody, FITC conjugated be utilized in antibody-drug conjugate (ADC) development?

TF represents an excellent target for antibody-drug conjugate development due to its rapid and efficient internalization properties. The development process typically involves:

• Selection of appropriate anti-TF antibody clone: Identify antibodies that efficiently induce inhibition of TF:FVIIa-dependent intracellular signaling, antibody-dependent cell-mediated cytotoxicity, and rapid target internalization, while minimally impacting TF procoagulant activity

• Conjugation strategy design: Methods include:

  • Direct conjugation to cytotoxic payloads such as monomethyl auristatin E (MMAE) or duostatin-3

  • Conjugation to loaded micellar nanoparticles containing drugs like epirubicin

• In vitro evaluation: Testing for:

  • Target-specific cytotoxicity (typically achieving IC50 values of 0.02-0.1 nM in TF-positive cells versus >100 nM in TF-negative cells, for a >5000-fold target selectivity)

  • Internalization efficiency

  • Lysosomal degradation rates

• In vivo assessment: Weekly intravenous administration protocols in xenograft models to determine:

  • Minimum effective dose (typically 0.3-1 mg/kg for MMAE conjugates)

  • Tolerability profile

  • Tumor suppression efficacy compared to conventional therapeutics

Research has demonstrated that TF-targeting ADCs have shown effective killing against tumor cell lines with variable levels of target expression and relative potency in reducing tumor growth compared with EGFR- and HER2-ADCs .

Why does TF demonstrate superior properties for ADC targeting compared to other receptors like EGFR and HER2?

TF exhibits several advantageous characteristics that make it particularly suitable for ADC development:

• Enhanced internalization efficiency: Both in the absence and presence of antibody, TF demonstrates more efficient internalization than EGFR and HER2

• Improved lysosomal targeting: Research has shown superior lysosomal targeting and degradation of TF compared to EGFR and HER2, which is critical for the release of cytotoxic payloads within target cancer cells

• Constant turnover rate: The constant turnover of TF on tumor cells makes this protein specifically suitable for an ADC approach, allowing for continuous delivery of cytotoxic agents to tumor cells

• Selective expression pattern: TF is frequently overexpressed in challenging tumor types like TNBC and pancreatic adenocarcinoma, which have limited therapeutic options, making it an attractive target for these difficult-to-treat cancers

Comparative studies have demonstrated that TF-ADC showed effective killing against tumor cell lines with variable levels of target expression and was relatively potent in reducing tumor growth compared with EGFR- and HER2-ADCs in xenograft models .

What methodological approaches can be used to assess TF antibody internalization kinetics?

To quantitatively evaluate TF antibody internalization, researchers can employ:

• Flow cytometry-based acid wash technique:

  • Label cells with FITC-conjugated TF antibody at 4°C

  • Incubate at 37°C for various time points (0-120 minutes)

  • Treat with acid wash buffer (0.2M acetic acid, 0.5M NaCl, pH 2.5) to remove surface-bound antibody

  • Analyze remaining intracellular fluorescence by flow cytometry

  • Calculate internalization rate as percentage of initial surface binding

• Confocal microscopy with co-localization markers:

  • Pulse-label cells with FITC-conjugated TF antibody

  • Chase at 37°C for various timepoints

  • Fix and co-stain with markers for early endosomes (EEA1), late endosomes (Rab7), or lysosomes (LAMP1)

  • Quantify co-localization coefficients to track intracellular trafficking

• pH-sensitive fluorescent dye quenching:

  • Label TF antibody with both pH-sensitive and pH-stable fluorophores

  • Monitor fluorescence ratio changes as antibody traffics to acidic compartments

  • Calculate internalization kinetics based on fluorescence quenching rates

Research has shown that TF demonstrates significantly faster internalization compared to other targeted receptors, with approximately 70-80% internalization within 60 minutes, compared to only 30-40% for receptors like EGFR under similar conditions .

What are common technical challenges when using TF Antibody, FITC conjugated and how can they be addressed?

When troubleshooting internalization studies specifically, researchers should validate that their TF antibody maintains the capacity to induce efficient inhibition of TF:FVIIa-dependent intracellular signaling while preserving rapid target internalization characteristics .

How can researchers optimize TF Antibody, FITC conjugated performance for specific experimental conditions?

For Flow Cytometry Optimization:

• Perform antibody titration to determine optimal concentration for your specific cell type

• Adjust cell density to ensure proper antibody-to-cell ratio (typically 1 × 10^6 cells/ml)

• Optimize incubation time and temperature based on internalization kinetics

• Include fluorescence-minus-one (FMO) controls to set accurate gates

• For fixed samples, evaluate different fixation protocols to preserve both antigen epitopes and FITC fluorescence

For Immunofluorescence Microscopy:

• Test different fixation methods (4% paraformaldehyde, methanol, or acetone) to determine optimal epitope preservation

• Evaluate permeabilization reagents (0.1-0.5% Triton X-100, 0.05-0.2% saponin) and their impact on signal intensity

• Incorporate nuclear counterstains that don't overlap with FITC spectrum (DAPI or Hoechst)

• Mount slides with anti-fade reagents containing DABCO or similar compounds to minimize photobleaching

• When imaging, begin with lower exposure settings and adjust incrementally to avoid photobleaching

For FACS-based Sorting Applications:

• Use viability dyes to exclude dead cells that can bind antibodies non-specifically

• Maintain samples at 4°C prior to sorting to minimize internalization

• Include DNase I (10-50 μg/ml) in buffers to prevent cell clumping

• Use preservative-free formulations for live cell sorting applications

• Optimize sorting gates based on positive control samples with known TF expression levels

Research groups have successfully applied these optimization approaches to achieve high affinity binding of anti-TF-conjugated constructs to TF-expressing cancer cell lines, with binding affinity comparable to that of unconjugated anti-human TF F(ab')2 fragments .

How can TF Antibody, FITC conjugated be utilized in the study of tumor microenvironment interactions?

The application of TF Antibody, FITC conjugated to study tumor microenvironment interactions represents an emerging research direction with several methodological approaches:

• Multi-parameter flow cytometry: Combining TF-FITC with markers for different cell populations allows researchers to characterize TF expression across various cell types in the tumor microenvironment, including cancer cells, endothelial cells, tumor-associated macrophages, and cancer-associated fibroblasts.

• Intravital microscopy: Using TF Antibody, FITC conjugated in combination with window chamber models enables real-time visualization of TF-expressing cells within the tumor microenvironment and their interactions with other cellular components.

• 3D organoid co-culture systems: Co-culturing TF-expressing cancer cells with stromal components and tracking with TF-FITC antibody allows for the study of TF's role in modulating cancer-stroma interactions.

• Spatial transcriptomics correlation: Combining TF-FITC immunofluorescence with spatial transcriptomics techniques provides insights into how TF expression correlates with specific gene expression programs in the tumor microenvironment.

Recent research suggests that TF expression influences tumor angiogenesis and stromal fibrosis, making these techniques valuable for studying how TF contributes to remodeling the tumor microenvironment .

What are the current experimental approaches for developing next-generation TF-targeted therapeutics?

Current experimental approaches for developing advanced TF-targeted therapeutics include:

• Bispecific antibody development: Engineering bispecific antibodies that simultaneously target TF and immune effector cells (T cells, NK cells) to enhance anti-tumor immune responses while blocking TF signaling.

• Novel payload conjugation strategies: Exploring new cytotoxic payloads beyond traditional auristatins, including:

  • DNA-damaging agents

  • RNA polymerase inhibitors

  • Immunomodulatory compounds

• Combination therapy optimization: Systematic evaluation of TF-targeted antibodies or ADCs in combination with:

  • Immune checkpoint inhibitors

  • Conventional chemotherapy

  • Radiation therapy

  • Anti-angiogenic agents

• TF-targeted nanoparticle systems: Development of more sophisticated delivery systems such as:

  • Anti-TF-conjugated polymeric micelles incorporating epirubicin or other anticancer agents

  • Liposomal formulations with anti-TF targeting

  • Biodegradable nanoparticles with controlled release properties

Research has demonstrated promising results with anti-TF-NC-6300, consisting of epirubicin-incorporating micelles conjugated with F(ab')2 fragments of anti-TF antibody. This approach achieved enhanced antitumor effects that were independent of tumor accumulation but dependent on selective intratumor localization and preferential internalization into high TF tumor cells .

How can researchers integrate TF Antibody, FITC conjugated into multiplexed imaging systems for advanced cancer diagnostics?

Integration of TF Antibody, FITC conjugated into multiplexed imaging systems requires careful consideration of several methodological aspects:

• Spectral unmixing protocols:

  • Implement linear unmixing algorithms to separate FITC signal from autofluorescence and other fluorophores

  • Acquire single-color controls for each fluorophore in the panel

  • Use reference spectra libraries to improve unmixing accuracy

• Panel design for multiplexed immunofluorescence:

  • Position FITC in appropriate channel based on expected TF expression level

  • Combine with markers for tumor cells (cytokeratins, EpCAM), immune cells (CD45, CD3, CD8), and vasculature (CD31)

  • Include proliferation markers (Ki67) and functional markers (cleaved caspase-3)

• Image acquisition optimization:

  • Standardize exposure times and laser power settings

  • Implement flat-field correction to account for illumination non-uniformity

  • Use appropriate z-stack sampling for volumetric analysis

• Quantitative image analysis workflows:

  • Develop cell segmentation algorithms for accurate identification of TF-positive cells

  • Implement spatial analysis tools to quantify TF-expressing cell distribution

  • Apply machine learning approaches for pattern recognition and phenotype classification

These advanced multiplexed imaging approaches can help researchers better understand the relationship between TF expression, tumor progression, and response to therapy, particularly in challenging cancers like triple-negative breast cancer and pancreatic adenocarcinoma where TF is frequently overexpressed .