TSPAN9 Antibody, FITC conjugated

What is TSPAN9 and what cellular functions does it regulate?

TSPAN9 (Tetraspanin-9) is a transmembrane protein belonging to the tetraspanin family that plays important roles in various cellular processes. In platelets, TSPAN9 is relatively specific to megakaryocytes and is a component of tetraspanin microdomains that include the collagen receptor GPVI and integrin α6β1, suggesting its involvement in regulating platelet function . Quantitative analysis has shown that TSPAN9 is expressed at approximately 2800 molecules per platelet cell surface, making it the third most abundant tetraspanin in platelets after CD9 and CD151 .

Beyond platelets, TSPAN9 has been implicated in cancer progression, particularly in osteosarcoma where it induces epithelial-mesenchymal transition (EMT) and promotes metastasis . Additionally, TSPAN9 plays a crucial role in viral entry, specifically for alphaviruses that require early endosomal fusion for infection . These diverse functions highlight TSPAN9 as an important regulatory protein in multiple biological contexts.

How does a FITC-conjugated TSPAN9 antibody differ from non-conjugated versions?

FITC (Fluorescein isothiocyanate)-conjugated TSPAN9 antibodies contain the fluorescent FITC molecule directly attached to the antibody, eliminating the need for secondary antibody detection steps. This conjugation provides several advantages in experimental workflows:

• Direct visualization: The FITC fluorophore (excitation ~495 nm, emission ~519 nm) allows immediate visualization of TSPAN9 in fixed or live cells without additional detection reagents.

• Reduced background: Elimination of secondary antibodies decreases non-specific binding and improves signal-to-noise ratios.

• Multiplexing capability: FITC can be combined with other fluorophores (e.g., PE, APC) in multi-color flow cytometry or immunofluorescence to simultaneously detect multiple proteins.

• Time efficiency: Direct conjugation reduces protocol steps and experimental time.

What experimental techniques are most compatible with FITC-conjugated TSPAN9 antibodies?

FITC-conjugated TSPAN9 antibodies are versatile reagents compatible with multiple experimental approaches:

• Flow cytometry: Ideal for quantitative analysis of TSPAN9 expression in cell populations, particularly in studies of platelets or cancer cells like osteosarcoma lines (HOS and U2OS) .

• Immunofluorescence microscopy: Effective for visualizing TSPAN9 cellular localization. Research has shown TSPAN9 localizes to plasma membranes and early/late endosomal compartments .

• Immunoprecipitation with fluorescence detection: FITC labeling enables visualization of protein complexes involving TSPAN9, such as its associations with integrin β1 .

• Live cell imaging: For monitoring dynamic TSPAN9 trafficking, particularly in endosomal compartments during processes like viral entry .

• FACS (Fluorescence-Activated Cell Sorting): To isolate TSPAN9-expressing cell populations for downstream applications.

For optimal results, each technique requires specific optimization of antibody concentration, incubation conditions, and appropriate controls, particularly considering FITC's sensitivity to photobleaching and pH changes.

What are the optimal fixation and permeabilization protocols for TSPAN9 immunostaining with FITC-conjugated antibodies?

The transmembrane nature of TSPAN9 necessitates careful consideration of fixation and permeabilization protocols to preserve epitope integrity while enabling antibody access. Based on successful TSPAN9 detection methods in published research, the following protocols are recommended:

For membrane TSPAN9 detection:

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

• Gentle permeabilization with 0.1% Triton X-100 (5 minutes at room temperature)

• Block with 5% normal serum (from species unrelated to the antibody)

• Incubate with FITC-conjugated TSPAN9 antibody (1:300 dilution recommended based on protocols used with unconjugated TSPAN9 antibodies)

For endosomal TSPAN9 detection:
When studying TSPAN9 in endosomal compartments, as in alphavirus entry studies , a milder permeabilization method preserves vesicular structures:

• Fix cells with 4% paraformaldehyde with 0.1% glutaraldehyde (15 minutes)

• Permeabilize with 0.1% saponin in PBS with 0.2% BSA

• Maintain 0.05% saponin in all subsequent washing and incubation steps

These protocols should be optimized for specific cell types and experimental conditions. Controls should include TSPAN9 knockdown cells (using verified siRNAs like siTspan9#1, siTspan9#2, or siTspan9#3) to confirm antibody specificity.

How can I validate the specificity of FITC-conjugated TSPAN9 antibodies in my experimental system?

Thorough validation of FITC-conjugated TSPAN9 antibodies is essential for generating reliable data. A comprehensive validation strategy should include:

• Genetic manipulation controls:

  • TSPAN9 knockdown: Perform siRNA-mediated knockdown using validated constructs (siTspan9#1, siTspan9#2, and siTspan9#3) and confirm reduced antibody staining

  • TSPAN9 overexpression: Transfect cells with TSPAN9 expression constructs (like those generated with FR-TSPAN9 fusion) and verify increased antibody staining

• Western blot correlation:

  • Compare flow cytometry or immunofluorescence results with Western blot analysis using validated anti-TSPAN9 antibodies (e.g., Abcam #ab106412, 1:300 dilution)

  • Confirm detection of the appropriate molecular weight band (~27-30 kDa, with variation due to glycosylation)

• N-glycosidase treatment:

  • Since TSPAN9 contains a predicted N-linked glycosylation site, treatment with N-glycosidase should reduce the apparent molecular weight to a single species in Western blot, confirming specificity

• Blocking peptide competition:

  • Pre-incubate the FITC-conjugated antibody with the immunizing peptide (such as the C-terminal QHIHRTGKKYDA peptide used for antibody generation)

  • Observe elimination of positive staining

• Multi-technique concordance:

  • Verify that expression patterns detected by the FITC-conjugated antibody match those observed with other detection methods and across different experimental systems

What is the recommended sample preparation protocol for detecting TSPAN9 in platelets using FITC-conjugated antibodies?

Platelets require specialized handling due to their sensitivity to activation. Based on established protocols for tetraspanin detection in platelets , the following procedure is recommended:

• Blood collection and platelet isolation:

  • Collect blood in acid-citrate-dextrose (ACD) anticoagulant

  • Prepare platelet-rich plasma (PRP) by centrifugation at 200g for 20 minutes

  • Wash platelets in modified Tyrode's buffer (134 mM NaCl, 2.9 mM KCl, 0.34 mM Na₂HPO₄, 12 mM NaHCO₃, 20 mM HEPES, 1 mM MgCl₂, 5 mM glucose, pH 7.3)

  • Include 0.05 U/ml apyrase and 10 ng/ml prostaglandin E₁ to prevent activation

• Fixation options:

  • For surface staining: Fix with 1% paraformaldehyde (10 minutes at room temperature)

  • For intracellular staining: Fix with 2% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

• Staining procedure:

  • Block with 2% BSA in PBS (30 minutes)

  • Incubate with FITC-conjugated TSPAN9 antibody (optimal concentration to be determined by titration)

  • Wash three times in PBS

  • For flow cytometry: Resuspend in PBS for immediate analysis

  • For microscopy: Mount in anti-fade mounting medium

• Analysis considerations:

  • Include CD42b (GPIbα) staining as a platelet marker

  • Set proper compensation if performing multicolor analysis with other tetraspanins such as CD9, CD151, or CD63

Based on quantitative studies, expect approximately 2,800 TSPAN9 molecules per platelet (compared to 49,000 CD9 molecules), which may require adequate sensitivity settings .

How can FITC-conjugated TSPAN9 antibodies be used to study tetraspanin microdomains in platelets?

Tetraspanin microdomains (TEMs) are specialized membrane structures that organize multiple proteins into functional complexes. Using FITC-conjugated TSPAN9 antibodies to study these structures in platelets requires sophisticated approaches:

• Super-resolution microscopy techniques:

  • STORM (Stochastic Optical Reconstruction Microscopy) or PALM (Photoactivated Localization Microscopy) can visualize TSPAN9-containing microdomains below the diffraction limit

  • Co-staining with other microdomain components like GPVI and integrin α6β1 (which have been shown to associate with TSPAN9) using spectrally distinct fluorophores

  • Quantify co-localization coefficients and nearest neighbor distances

• Proximity ligation assay (PLA):

  • Combine FITC-conjugated TSPAN9 antibody with antibodies against potential interaction partners

  • PLA signals will indicate proteins in close proximity (<40 nm), suggesting microdomain co-localization

• Co-immunoprecipitation with fluorescence detection:

  • Use non-ionic detergents like 1% Nonidet P40 to preserve tetraspanin-tetraspanin interactions

  • More stringent detergents like 1% dodecylmaltoside can be used to disrupt these interactions for control comparisons

  • Direct visualization of FITC fluorescence in precipitated complexes

• FRET (Förster Resonance Energy Transfer) analysis:

  • Pair FITC-TSPAN9 antibody with antibodies against potential partners labeled with compatible FRET acceptors

  • FRET occurs only when proteins are within ~10 nm, providing evidence for direct molecular interactions

A practical experimental approach would involve comparing TSPAN9 microdomain composition in resting platelets versus activated platelets (with collagen or thrombin), enabling dynamic understanding of how these microdomains reorganize during platelet activation and aggregation.

What approaches can be used to study TSPAN9's role in EMT and cancer metastasis using FITC-conjugated antibodies?

TSPAN9 has been implicated in promoting epithelial-mesenchymal transition (EMT) and metastasis in osteosarcoma . FITC-conjugated TSPAN9 antibodies can be valuable tools in studying these processes:

• Dual immunofluorescence for TSPAN9 and EMT markers:

  • Co-stain for TSPAN9 (FITC-conjugated antibody) and EMT markers (using antibodies with distinct fluorophores)

  • Analyze correlations between TSPAN9 expression levels and EMT markers such as:

    • N-Cadherin (decreased cell adhesion)

    • Vimentin (mesenchymal cytoskeletal marker)

    • Fibronectin (FN1, extracellular matrix protein)

    • Snai1 (EMT transcription factor)

    • β-Catenin (Wnt pathway component)

• Live-cell imaging of cell migration:

  • Transfect cells with fluorescently-tagged TSPAN9 constructs (such as FusionRed-TSPAN9)

  • Perform time-lapse imaging to correlate TSPAN9 localization with leading edges of migrating cells

  • Quantify migration parameters (velocity, directionality, persistence)

• Flow cytometry analysis of circulating tumor cells:

  • Use FITC-conjugated TSPAN9 antibody to identify potential metastasis-initiating cells

  • Combine with other markers to create a phenotypic profile of metastatic cells

  • Sort cells for further functional characterization

• Intravital imaging in mouse models:

  • Inject FITC-conjugated TSPAN9 antibody to visualize tumor cells with high TSPAN9 expression in vivo

  • Track cell movement, extravasation, and colonization at metastatic sites

  • Correlate with histopathological findings from fixed tissues

Research has shown that TSPAN9 knockdown significantly impairs osteosarcoma metastasis in vivo, with fewer pulmonary metastatic nodules compared to control groups (see table below) :

How can FITC-conjugated TSPAN9 antibodies help elucidate the role of TSPAN9 in viral entry pathways?

TSPAN9 has been identified as a factor in alphavirus entry and early endosome fusion events . FITC-conjugated TSPAN9 antibodies offer several sophisticated approaches to study this process:

• High-resolution colocalization analysis:

  • Track the colocalization of FITC-labeled TSPAN9 with fluorescently labeled viral particles

  • Quantify colocalization coefficients during different stages of viral entry

  • Analyze the temporal dynamics of TSPAN9-virus association in early endosomes

• Live-cell imaging of viral trafficking:

  • Combine FITC-conjugated TSPAN9 antibodies with fluorescently labeled viruses

  • Use spinning disk confocal microscopy for rapid acquisition of viral trafficking events

  • Measure parameters such as:

    • Time of TSPAN9-virus colocalization

    • Endosome maturation rates in TSPAN9-positive compartments

    • Fusion events within TSPAN9-positive endosomes

• TSPAN9 depletion/reconstitution studies:

  • Create TSPAN9 knockdown cells using validated siRNAs

  • Reconstitute with wild-type or mutant TSPAN9 constructs

  • Use FITC-conjugated antibodies to confirm expression levels

  • Correlate expression with viral infection efficiency

Research has shown that TSPAN9 depletion differentially affects viruses based on their fusion pH requirements :

These findings suggest TSPAN9 specifically modulates early endosome compartment fusion permissiveness.

How can I address high background when using FITC-conjugated TSPAN9 antibodies in immunofluorescence?

High background is a common challenge with fluorescence microscopy that can obscure specific TSPAN9 staining. Several strategies can address this issue:

• Optimize blocking conditions:

  • Increase blocking time (try 1-2 hours)

  • Test different blocking agents (5% normal serum, 3% BSA, commercial blocking solutions)

  • Add 0.1-0.3% Triton X-100 to blocking solution to reduce hydrophobic interactions

• Antibody titration:

  • Perform a dilution series (starting from 1:100 to 1:1000)

  • Compare signal-to-noise ratio at each dilution

  • Select the dilution with optimal specific signal and minimal background

• Reduce autofluorescence:

  • Pre-treat samples with 0.1% sodium borohydride (10 minutes) to reduce aldehyde-induced autofluorescence

  • For tissues with high autofluorescence (e.g., bone sections in osteosarcoma studies), treat with 0.1-1% Sudan Black B in 70% ethanol after antibody incubation

  • Include an additional 10mM copper sulfate treatment in 50mM ammonium acetate buffer (pH 5.0) for tissues with high elastin/collagen content

• Washing optimization:

  • Increase wash duration and number of washes (5 washes, 5 minutes each)

  • Add 0.05% Tween-20 to wash buffer to reduce non-specific binding

  • Use TBS instead of PBS if phosphate interactions are suspected to contribute to background

• Controls and troubleshooting:

  • Always include a negative control (secondary antibody only or isotype control)

  • Use TSPAN9 knockdown samples as biological negative controls

  • Consider using biotinylated primary antibodies with streptavidin-conjugated fluorophores for signal amplification while maintaining specificity

For particularly challenging samples, consider photobleaching the sample briefly before imaging to reduce autofluorescence, then image FITC signal (which will recover more quickly than autofluorescence).

What are the potential pitfalls in quantifying TSPAN9 expression levels by flow cytometry with FITC-conjugated antibodies?

Accurate quantification of TSPAN9 by flow cytometry requires addressing several technical considerations:

• FITC-specific issues:

  • FITC is pH-sensitive: Maintain consistent pH in all buffers (pH 7.4)

  • Photobleaching: Minimize exposure to light during sample preparation

  • Spectral overlap: Properly compensate when using multiple fluorophores

  • Quenching: Avoid including proteins that can quench FITC (e.g., excess heme proteins)

• Antibody saturation:

  • Perform titration experiments to ensure saturation binding

  • Create a saturation curve and select a concentration on the plateau

• Standardization for absolute quantification:

  • Use calibration beads with known quantities of fluorophore (e.g., MESF beads)

  • Follow the methodology used for CD9 quantification with the Platelet Calibrator Kit

  • Create standard curves with recombinant TSPAN9 proteins

• Membrane permeabilization considerations:

  • TSPAN9 has both surface and intracellular pools; decide whether to measure total or surface expression

  • If measuring total expression, ensure consistent permeabilization across samples

  • For surface-only measurements, avoid permeabilization reagents

• Validation approaches:

  • Compare flow cytometry results with Western blot quantification

  • Use the quantitative Western blotting method as described for tetraspanin quantification in platelets

A detailed example of quantitative analysis from TSPAN9 research in platelets showed that the relative expression levels of tetraspanins were CD9:CD151:Tspan9:CD63 = 100:14:6:2, with CD9 expressed at 49,000 copies per platelet, suggesting approximately 2,800 TSPAN9 molecules per platelet . Similar approaches can be adapted for TSPAN9 quantification in other cell types.

How should I interpret changes in TSPAN9 localization versus expression levels in my experimental system?

Distinguishing between changes in TSPAN9 expression and subcellular localization is crucial for proper biological interpretation. The following methodological approaches can help:

• Combined analysis strategies:

  • Western blotting: Quantifies total TSPAN9 protein levels

  • Flow cytometry: Measures per-cell expression distribution

  • Immunofluorescence: Reveals subcellular localization patterns

  • Cell fractionation: Separates membrane, cytosolic, and endosomal populations

• Specific markers for colocalization studies:

  • Plasma membrane: Na⁺/K⁺-ATPase

  • Early endosomes: EEA1, Rab5

  • Late endosomes: LAMP1, LAMP2, CD63 (note that TSPAN9 depletion affects LAMP1 and CD63 levels)

  • Calculate Pearson's correlation coefficients for quantitative assessment

• Time-course experiments:

  • Monitor TSPAN9 redistribution following stimuli (e.g., viral infection, EMT induction)

  • Distinguish acute relocalization (minutes to hours) from expression changes (hours to days)

• Functional correlations:

  • For platelet studies: Correlate TSPAN9 localization with activation states

  • For cancer studies: Compare localization in epithelial versus mesenchymal phenotypes

  • For viral entry: Track localization during infection progression

• Interpretation framework:

In osteosarcoma cells, research has demonstrated that TSPAN9 not only shows increased expression (fold-change = 2.131, P = 0.0037 in GSE12865 dataset) , but also undergoes redistribution to membrane protrusions during EMT, suggesting both transcriptional regulation and functional relocalization contribute to its pro-metastatic effects.

How might FITC-conjugated TSPAN9 antibodies be used in combination with other emerging technologies for tetraspanin research?

The integration of FITC-conjugated TSPAN9 antibodies with cutting-edge technologies opens new research frontiers:

• Single-molecule localization microscopy:

  • Use photoconvertible FITC derivatives for PALM imaging

  • Map precise nanoscale organization of TSPAN9 within tetraspanin-enriched microdomains (TEMs)

  • Determine stoichiometry of TSPAN9 in protein complexes

• Expansion microscopy:

  • Physically expand samples to achieve super-resolution with standard confocal microscopy

  • Visualize TSPAN9 interactions with binding partners at nanoscale resolution

  • Map endosomal TSPAN9 distribution during viral entry with unprecedented detail

• Spatial transcriptomics with protein detection:

  • Combine FITC-TSPAN9 antibody staining with in situ RNA sequencing

  • Correlate TSPAN9 protein expression with transcriptional programs in tissue microenvironments

  • Map cell states during EMT progression in tumor samples

• Organoid and microfluidic technologies:

  • Monitor TSPAN9 expression and localization in 3D organoid cultures

  • Study dynamic TSPAN9 redistribution during cancer invasion in microfluidic devices

  • Visualize platelet TSPAN9 under flow conditions mimicking vascular environments

• CRISPR-based imaging:

  • Combine CRISPR-mediated TSPAN9 tagging with FITC-conjugated antibodies

  • Create endogenously tagged TSPAN9 cell lines for live imaging

  • Study dynamic regulation without overexpression artifacts

These emerging approaches will provide unprecedented insights into TSPAN9 biology across multiple research contexts, from basic tetraspanin microdomain organization to complex disease processes.

What considerations are important when designing experiments to resolve contradictory findings about TSPAN9 function?

Contradictory findings regarding TSPAN9 function (e.g., anti-oncogenic in gastric cancer vs. pro-metastatic in osteosarcoma ) necessitate carefully designed experiments:

• Cell type-specific effects:

  • Compare TSPAN9 expression and function across multiple cell types using identical methodologies

  • Use FITC-conjugated antibodies to quantify expression in different tissues

  • Analyze co-expression patterns with potential interaction partners

• Context-dependent protein interactions:

  • Perform comparative co-immunoprecipitation studies across cell types

  • Map TSPAN9 interactome using proximity labeling techniques

  • Determine if TSPAN9 associates with different partners in different contexts

• Signaling pathway analysis:

  • Investigate whether TSPAN9 activates different downstream pathways in different cell types

  • Compare effects on FAK-Ras-ERK1/2 signaling across contexts

  • Use phospho-specific antibodies to map signaling events

• Isoform-specific functions:

  • Design experiments to detect potential TSPAN9 splice variants

  • Analyze post-translational modifications across tissues (e.g., differential glycosylation patterns)

  • Create isoform-specific constructs for rescue experiments

• Experimental design principles:

  • Use multiple independent approaches to verify findings

  • Include positive and negative controls in each experiment

  • Standardize experimental conditions across comparative studies

  • Employ genetic manipulation to confirm antibody specificity

A systematic approach might utilize FITC-conjugated TSPAN9 antibodies to perform quantitative expression analysis across a tissue panel, followed by co-immunoprecipitation studies to identify context-specific interaction partners, thereby explaining seemingly contradictory functions in different cellular environments.

How can researchers effectively combine genetic approaches with FITC-conjugated TSPAN9 antibodies to elucidate novel functions?

Integrating genetic manipulation with antibody-based detection creates powerful experimental paradigms for TSPAN9 research:

• CRISPR/Cas9 genome editing strategies:

  • Generate TSPAN9 knockout cell lines for antibody validation

  • Create domain-specific mutations to map functional regions

  • Introduce epitope tags for pulldown experiments

  • Use FITC-conjugated antibodies to verify editing efficiency and phenotypic consequences

• RNA interference approaches:

  • Implement siRNA knockdown (using validated constructs like siTspan9#1, #2, #3)

  • Establish stable shRNA expression systems for long-term studies

  • Use FITC-conjugated antibodies for quantitative assessment of knockdown efficiency

  • Perform rescue experiments with siRNA-resistant constructs

• Overexpression systems:

  • Create inducible TSPAN9 expression systems

  • Develop tissue-specific expression models for in vivo studies

  • Generate fluorescent protein fusions (like FR-TSPAN9) for dynamic imaging

  • Use domain swapping to create chimeric proteins

• Animal models:

  • Develop TSPAN9 knockout or conditional knockout mice

  • Create tissue-specific TSPAN9 transgenic models

  • Use FITC-conjugated antibodies for tissue section analysis

  • Implement intravital imaging in appropriate models

• Functional readouts:

  • For cancer studies: invasion assays, metastasis models, EMT marker analysis

  • For platelet studies: aggregation assays, thrombus formation, bleeding time

  • For viral entry: infection efficiency, virus trafficking, endosome fusion

A comprehensive experimental approach might involve:

• CRISPR/Cas9-mediated generation of TSPAN9 domain mutants

• Quantification of expression using FITC-conjugated antibodies

• Colocalization analysis with tetraspanin microdomain components

• Functional assays relevant to the biological context

• Rescue experiments to confirm specificity

This multi-faceted strategy would provide robust evidence for novel TSPAN9 functions while controlling for potential confounding factors.