CD81 Antibody, FITC conjugated

What is CD81 and what role does it play in cellular biology?

CD81 (also known as TAPA-1, target of anti-proliferative antibody-1) is a 25-26 kDa transmembrane protein belonging to the tetraspanin family. It plays critical roles in various cellular processes including cell adhesion, morphology regulation, activation, proliferation, and differentiation of immune cells. CD81 forms complexes with other tetraspanin proteins, integrins, coreceptors, and MHC class I and II molecules, functioning as an important mediator in cellular signaling pathways. In muscle tissue, CD81 promotes cell fusion and supports myotube maintenance. Additionally, CD81 has been identified as a receptor for the hepatitis C virus, making it a target of interest in infectious disease research . The broad distribution of CD81 across cell types makes it an important marker for characterizing various cell populations in immunological research.

Which cell types express CD81 and can be detected with FITC-conjugated antibodies?

CD81 has a remarkably broad cellular distribution compared to many CD markers. According to the research data, CD81 is expressed on the majority of nucleated cells, but with notable expression patterns in the hematopoietic system. Specifically, CD81 is highly expressed on:

• B and T lymphocytes

• NK cells

• Thymocytes

• Eosinophils

• Germinal center follicular dendritic cells

• Variable expression on monocytes

Importantly, CD81 is not expressed on neutrophils, platelets, or erythrocytes . This expression profile makes CD81-FITC antibodies particularly useful for identifying and characterizing lymphoid populations, especially germinal center B cells which show enhanced expression levels . The broad expression pattern requires careful gating strategies when using CD81-FITC in flow cytometry panels targeting specific cell populations.

What are the optimal storage conditions for CD81-FITC antibodies?

Proper storage is crucial for maintaining the reactivity and fluorescence intensity of CD81-FITC conjugated antibodies. Based on manufacturer recommendations, these antibodies should be:

• Stored at 2-8°C (refrigerated, not frozen)

• Protected from prolonged exposure to light to prevent photobleaching of the FITC fluorochrome

• Never frozen, as freezing can damage the antibody structure and fluorochrome conjugation

• Kept in their original stabilizing formulation, typically phosphate buffered saline (PBS), pH 7.4, with 15 mM sodium azide

When properly stored, CD81-FITC antibodies typically maintain their performance for at least 12 months from the date of manufacture. Repeated freeze-thaw cycles and exposure to bright light should be strictly avoided as these conditions significantly reduce antibody performance and fluorescence intensity.

How does FITC conjugation affect CD81 antibody performance?

FITC conjugation provides a convenient means for detecting CD81 through flow cytometry, but researchers should understand how this modification impacts antibody characteristics. The FITC fluorochrome has an excitation maximum at approximately 490nm and emission maximum around 525nm, making it compatible with the blue laser (488nm) found in most flow cytometers .

When antibodies are conjugated with FITC, several factors should be considered:

• The conjugation process can occasionally affect the antibody's binding affinity or specificity

• FITC has a relatively lower brightness compared to newer fluorochromes like PE or APC

• FITC is susceptible to photobleaching and its fluorescence is pH-sensitive

• FITC emission has significant overlap with cellular autofluorescence, potentially affecting sensitivity in cells with high autofluorescence

Most manufacturers optimize conjugation conditions to minimize these effects. For example, the M38 clone is conjugated with FITC under optimum conditions, and unconjugated antibody and free fluorochrome are removed by size-exclusion chromatography to ensure quality and performance .

What is the recommended protocol for staining cells with CD81-FITC antibodies?

For optimal results when using CD81-FITC antibodies in flow cytometry, researchers should follow this standardized protocol:

• Prepare cell suspension at appropriate concentration (typically 10^6 cells per test)

• For whole blood applications: Use 20 μl reagent per 100 μl of whole blood

• For cell suspensions: Use 20 μl reagent per 10^6 cells in suspension

• Incubate for 20-30 minutes at 2-8°C in the dark

• If using whole blood, lyse red blood cells using an appropriate lysis solution

• Wash cells twice with PBS containing 2% FBS to remove unbound antibody

• Resuspend in an appropriate buffer for flow cytometric analysis

• Analyze using a flow cytometer with a blue (488 nm) excitation laser

When working with CD81-FITC antibodies, it's important to note that a typical vial (2 ml) contains sufficient reagent for approximately 100 tests when using the recommended volumes. For multicolor applications, adjustments to compensation settings may be necessary to account for spectral overlap with other fluorochromes.

What controls are essential when using CD81-FITC antibodies in flow cytometry?

When designing experiments with CD81-FITC antibodies, incorporating proper controls is crucial for reliable data interpretation:

• Isotype Control: Include a mouse IgG1-FITC isotype control at the same concentration as the CD81-FITC antibody to assess non-specific binding and properly set gates

• Unstained Control: Essential for determining cellular autofluorescence and setting negative population boundaries

• Single-Color Controls: Required for proper compensation when performing multicolor flow cytometry

• Fluorescence Minus One (FMO) Controls: Include all fluorochromes except CD81-FITC to precisely determine the boundary between positive and negative populations

• Positive Control: Include a sample type known to express CD81 (e.g., lymphocytes) to confirm antibody performance

• Negative Control: Where possible, include cells known not to express CD81 (e.g., erythrocytes) as a biological negative control

Proper implementation of these controls ensures accurate identification of CD81-positive populations and minimizes the risk of false positive or negative results due to technical factors.

How can CD81-FITC antibodies be incorporated into multicolor flow cytometry panels?

Integrating CD81-FITC antibodies into multicolor panels requires careful consideration of spectral overlap and marker co-expression patterns:

• Panel Design Considerations:

  • FITC exhibits spectral overlap with PE, which must be compensated for

  • Position CD81-FITC on the FITC channel (typically FL1 on many cytometers)

  • Combine with markers on non-overlapping channels for easier analysis

  • Consider using brighter fluorochromes for rare or low-expression markers

• Recommended Marker Combinations:

  • For B cell characterization: CD81-FITC, CD19-PE, CD20-APC

  • For T cell studies: CD81-FITC, CD3-PerCP, CD4-APC, CD8-PE-Cy7

  • For studying tetraspanin networks: CD81-FITC, CD9-PE, CD63-APC

• Compensation Strategy:

  • Always prepare single-stained controls for each fluorochrome

  • Use cells rather than beads for compensation when possible

  • Consider computational tools for complex panels with significant spectral overlap

When selecting clones for multicolor panels, researchers should be aware that different CD81-FITC antibody clones exist, including M38, JS-81, and Eat2 . Clone selection should be based on the specific application and experimental needs, with consideration for potential clone-specific differences in epitope recognition.

How should I determine the optimal concentration of CD81-FITC antibody for my experiment?

Determining the optimal concentration of CD81-FITC antibody is crucial for obtaining clear separation between positive and negative populations while minimizing background staining:

• Titration Experiment:

  • Prepare serial dilutions of the antibody (typically 2-fold dilutions)

  • Start with the manufacturer's recommended concentration (typically 20 μl per 10^6 cells)

  • Include both higher and lower concentrations

  • Stain identical aliquots of your target cells with each dilution

• Analysis Approach:

  • Calculate the staining index for each concentration using the formula:
    SI = (MFI positive - MFI negative) / (2 × SD of negative)

  • Plot the staining index against antibody concentration

  • Select the concentration that gives the highest staining index

• Concentration Considerations:

  • Higher concentrations may increase sensitivity but can also increase non-specific binding

  • Lower concentrations may reduce background but could miss cells with low expression

  • The optimal concentration may vary between different sample types and preparation methods

While manufacturers typically recommend 20 μl of reagent per 100 μl of whole blood or 10^6 cells , this should be validated for each specific experimental system to ensure optimal performance.

How can CD81-FITC antibodies be used to investigate hepatitis C virus interactions?

CD81 has been identified as a receptor for the hepatitis C virus (HCV), making CD81-FITC antibodies valuable tools in studying HCV-host interactions:

• Receptor Binding Studies:

  • CD81-FITC antibodies can be used to quantify CD81 expression levels on hepatocytes or hepatocyte cell lines

  • Competitive binding assays using CD81-FITC and HCV envelope protein E2 can help characterize binding domains

  • Flow cytometry with CD81-FITC can identify cells susceptible to HCV infection based on receptor expression

• Infection Model Applications:

  • Monitor changes in CD81 expression during HCV infection progression

  • Assess effects of antiviral treatments on CD81 expression and distribution

  • Study CD81 co-localization with other HCV entry factors using multi-parameter analysis

• Methodological Approach:

  • Combine CD81-FITC staining with intracellular HCV antigen detection

  • Use CD81-FITC in receptor blocking experiments to confirm specificity

  • Employ CD81-FITC to sort cells based on receptor expression levels for downstream analysis

This application leverages the specific binding of CD81-FITC antibodies to the extracellular epitope of CD81, which is the same region involved in HCV E2 protein interaction . The fluorescent labeling allows researchers to visualize and quantify the receptor availability on cell surfaces, correlating this with susceptibility to viral infection.

What are the considerations when using CD81-FITC for studying tetraspanin-enriched microdomains?

Tetraspanin-enriched microdomains (TEMs) are specialized membrane structures where CD81 plays a critical organizational role:

• Experimental Design for TEM Analysis:

  • CD81-FITC can be used to identify and quantify TEMs on cell surfaces

  • Co-staining with antibodies against other tetraspanins (CD9, CD63, CD82) helps characterize TEM composition

  • Combining CD81-FITC with membrane fluidity probes provides insights into TEM dynamics

• Technical Considerations:

  • Gentle fixation protocols are essential as harsh fixatives can disrupt TEMs

  • Avoid detergents in buffers when studying intact TEMs

  • Consider using advanced techniques like imaging flow cytometry for visualization of TEMs

• Analytical Approaches:

  • Measure co-localization coefficients between CD81-FITC and other TEM markers

  • Analyze clustering patterns using high-resolution imaging techniques

  • Quantify fluorescence intensity ratios to assess relative abundance of CD81 in TEMs

CD81-FITC antibodies provide a means to study the role of CD81 in organizing these microdomains, which influence numerous cellular processes including signal transduction, cell adhesion, and membrane compartmentalization . Understanding these structures has implications for immunology, cancer biology, and infectious disease research.

How does CD81 expression correlate with B cell activation states?

CD81 expression on B cells changes dynamically during activation and differentiation, making CD81-FITC antibodies valuable for characterizing B cell functional states:

• Expression Patterns During B Cell Development:

  B Cell Stage	CD81 Expression Level	Associated Markers
  Naïve B cell	Moderate	CD19+, IgD+, CD27-
  Germinal center B cell	High	CD19+, CD38+, CD27+
  Memory B cell	Moderate to High	CD19+, CD27+, IgD-
  Plasma cell	Decreased	CD19low, CD38high, CD138+

• Functional Correlations:

  • Increased CD81 expression correlates with B cell activation and proliferation

  • CD81 clustering is associated with BCR signaling activation

  • CD81 modulates the threshold for B cell activation through associations with CD19/CD21 complex

• Research Applications:

  • Use CD81-FITC to monitor B cell activation states in response to stimuli

  • Track germinal center reactions through CD81 expression changes

  • Study antibody responses by characterizing CD81 expression on responding B cells

CD81-FITC staining intensity provides valuable insights into B cell functionality, as CD81 influences B cell receptor signaling thresholds and plays a role in germinal center organization . Multiparameter analysis combining CD81-FITC with other B cell markers enables detailed characterization of B cell subsets and their functional states during immune responses.

What role does CD81 play in immunological synapse formation?

CD81's involvement in immunological synapse formation makes CD81-FITC antibodies useful for studying T cell-APC interactions:

• CD81 Function in Immunological Synapses:

  • Facilitates spatial organization of receptors and signaling molecules

  • Mediates stabilization of T cell-APC contacts

  • Modulates signal strength and duration at the synapse

  • Influences cytoskeletal reorganization during synapse formation

• Experimental Approaches Using CD81-FITC:

  • Live cell imaging of CD81-FITC distribution during synapse formation

  • Quantify CD81 enrichment at contact zones between T cells and APCs

  • Correlate CD81 localization with functional outcomes of T cell activation

• Advanced Applications:

  • Combined with phospho-flow cytometry to link CD81 organization to signaling events

  • Used in super-resolution microscopy to visualize nanoscale organization of CD81 in the synapse

  • Employed in FRET studies to examine protein-protein interactions involving CD81

CD81-FITC antibodies allow researchers to visualize and quantify the dynamic redistribution of CD81 during immunological synapse formation, providing insights into T cell activation mechanisms . This application area represents an intersection between immunology and cellular biology, with implications for understanding T cell-mediated immune responses.

How can I address weak or variable CD81-FITC staining in my samples?

Inconsistent or weak CD81-FITC staining can arise from various technical or biological factors:

• Common Causes and Solutions:

  • Antibody Degradation: Store protected from light at 2-8°C; avoid freeze-thaw cycles

  • Cell Viability Issues: Include viability dye to exclude dead cells; optimize sample preparation

  • Epitope Masking: Try alternative fixation methods; stain before fixation if possible

  • Insufficient Antibody: Perform titration experiments to determine optimal concentration

  • Excessive Washing: Reduce wash steps or adjust wash buffer composition

• Cell Type-Specific Considerations:

  • Some cell types naturally express lower levels of CD81

  • Activated cells may show altered CD81 expression or distribution

  • Sample processing can affect membrane protein integrity differently across cell types

• Technical Optimization Strategies:

  • Extend incubation time (up to 45 minutes) at lower temperatures

  • Use fresh samples whenever possible

  • Consider alternative CD81 antibody clones if consistent issues persist

  • Try saponin-based permeabilization for detecting intracellular CD81 pools

Implementing these approaches systematically can help resolve staining issues and ensure consistent CD81-FITC detection across experiments.

What might cause false positive or negative results when using CD81-FITC antibodies?

Understanding potential sources of error is critical for accurate interpretation of CD81-FITC staining:

• Causes of False Positive Results:

  • Insufficient washing leading to nonspecific antibody retention

  • Autofluorescence, particularly in the FITC channel (addressed through proper controls)

  • Spectral overlap from other fluorochromes (requires proper compensation)

  • Fc receptor binding (can be blocked with Fc receptor blocking reagents)

  • Damaged cells with increased membrane permeability allowing non-specific binding

• Causes of False Negative Results:

  • Epitope masking due to inappropriate fixation or permeabilization

  • CD81 downregulation in certain physiological or pathological conditions

  • Epitope competition with endogenous ligands or other proteins

  • Antibody degradation due to improper storage

  • Insufficient antibody concentration for detection

• Validation Approaches:

  • Use alternative CD81 antibody clones targeting different epitopes

  • Correlate flow cytometry findings with other detection methods

  • Include known positive and negative control samples in each experiment

  • Perform blocking experiments to confirm specificity

Careful experimental design incorporating appropriate controls and standardized protocols minimizes the risk of both false positive and negative results when working with CD81-FITC antibodies .

How do I interpret CD81 expression levels in different cell populations?

Interpreting CD81 expression patterns requires understanding its biological context across cell types:

• Expression Level Interpretation:

  • CD81 is expressed on most nucleated cells but at varying levels

  • Higher expression on B cells, particularly germinal center B cells

  • Variable expression on monocytes requires careful gating

  • Absence on neutrophils, platelets, and erythrocytes serves as internal negative control

• Analytical Approaches:

  • Use median fluorescence intensity (MFI) rather than percent positive for quantitative comparisons

  • Apply standardization methods (such as MESF beads) for cross-experiment comparisons

  • Consider relative expression ratios between different markers rather than absolute values

  • Use density plots rather than histograms for better visualization of co-expression patterns

• Biological Context Considerations:

  • CD81 expression can change with cell activation state

  • Expression patterns may differ between healthy and disease states

  • Species differences in expression patterns exist (validated for human, rabbit, and cat)

  • Consider tissue-specific regulation of CD81 expression

Understanding these nuances allows researchers to properly interpret CD81 expression data in the context of their specific research questions.

What approaches help distinguish specific CD81-FITC signals from autofluorescence?

Cellular autofluorescence can interfere with FITC detection, particularly in certain cell types:

• Technical Strategies:

  • Implement proper unstained and isotype controls

  • Use Fluorescence Minus One (FMO) controls for precise gating

  • Consider alternative fluorochromes for cells with high autofluorescence

  • Apply spectral unmixing algorithms on advanced flow cytometers

• Sample Preparation Considerations:

  • Minimize fixation-induced autofluorescence by using aldehyde-free fixatives

  • Quench autofluorescence with reagents like trypan blue or crystal violet

  • Reduce culture media exposure time before staining

  • Optimize red cell lysis protocols to minimize debris

• Analytical Approaches:

  • Use multiparameter analysis to better define positive populations

  • Implement autofluorescence extraction channels in analysis

  • Apply fluorescence subtraction algorithms during analysis

  • Consider alternative analysis methods like fluorescence ratios

These approaches are particularly important when working with cells known to have high autofluorescence, such as macrophages, dendritic cells, or cells from certain tissues like lung or liver.

How do different clones of CD81-FITC antibodies compare in performance?

Several CD81 antibody clones are available as FITC conjugates, each with distinct characteristics:

• Clone Comparison:

  Clone	Epitope Location	Species Reactivity	Optimal Application
  M38	Extracellular	Human, Rabbit, Cat	Flow cytometry
  JS-81	Extracellular	Human	Flow cytometry, functional studies
  Eat2	Extracellular	Human	General-purpose detection

• Performance Differences:

  • Clone M38 shows broad species cross-reactivity, making it versatile for comparative studies

  • JS-81 has been extensively validated for functional studies investigating CD81's role in cellular processes

  • Different clones may exhibit varied staining intensities depending on the epitope accessibility

• Selection Criteria:

  • Choose based on validated reactivity with your species of interest

  • Consider epitope location for functional studies

  • Review published literature using specific clones for your application

  • Evaluate performance in your specific experimental system through direct comparison

Understanding these differences helps researchers select the most appropriate CD81-FITC antibody clone for their specific research applications.

What are the considerations when using CD81-FITC antibodies across different species?

While CD81 is conserved across species, there are important considerations for cross-species applications:

• Validated Species Reactivity:

  • Clone M38 has confirmed reactivity with human, rabbit, and cat CD81

  • Other clones may have more limited species reactivity

  • Even with validated cross-reactivity, epitope recognition strength may vary across species

• Experimental Design Considerations:

  • Include appropriate species-specific positive controls

  • Validate antibody performance in each species before extensive studies

  • Consider species-specific differences in CD81 expression patterns

  • Adjust antibody concentrations when changing species (may require re-titration)

• Alternative Approaches:

  • For non-validated species, consider testing multiple CD81 antibody clones

  • Molecular validation (e.g., western blot) can complement flow cytometry data

  • Species-specific secondary detection systems may enhance sensitivity

These considerations are particularly important for veterinary research or comparative immunology studies where multiple species are being investigated simultaneously.

How does fixation affect CD81 epitope recognition by FITC-conjugated antibodies?

Fixation procedures can significantly impact CD81 detection with FITC-conjugated antibodies:

• Fixation Effects on CD81 Epitopes:

  • Paraformaldehyde fixation generally preserves CD81 epitopes but may reduce staining intensity

  • Alcohol-based fixatives can disrupt membrane protein organization, affecting tetraspanin detection

  • Glutaraldehyde fixation often masks CD81 epitopes, reducing antibody binding

• Recommended Fixation Protocols:

  • For flow cytometry: Use mild fixation (0.5-2% paraformaldehyde) after staining

  • For preservation of tetraspanin microdomains: Consider alternative fixatives like glyoxal

  • When possible, perform staining on live cells and fix after antibody binding

• Optimization Strategies:

  • Test multiple fixation conditions with appropriate controls

  • Consider alternative fixatives for specialized applications

  • Implement antigen retrieval steps if fixed samples show reduced staining

  • Adjust antibody concentration to compensate for fixation-induced sensitivity changes

Understanding these interactions helps researchers design protocols that maintain CD81 epitope accessibility while achieving necessary sample preservation for their specific applications.

What are the advantages and limitations of CD81-FITC compared to other tetraspanin markers?

CD81-FITC offers specific advantages and limitations compared to other tetraspanin family markers:

• Comparative Advantages:

  • CD81 has broader cellular distribution than many other tetraspanins

  • Strong expression on lymphocytes makes it excellent for immune cell phenotyping

  • Well-characterized involvement in HCV binding provides specific research applications

  • Available antibody clones show good species cross-reactivity

• Limitations:

  • Broad expression pattern can make it less specific for certain cell identification

  • FITC conjugation limits sensitivity compared to brighter fluorochromes

  • Functional redundancy with other tetraspanins can complicate interpretation

  • Some epitopes may be masked in tetraspanin-enriched microdomains

• Complementary Tetraspanin Markers:

  • CD9: Often used in conjunction with CD81 for exosome studies

  • CD63: Provides insights into endosomal-tetraspanin interactions

  • CD82: Useful for studying tetraspanin involvement in tumor suppression

Researchers should consider these comparative aspects when selecting CD81-FITC or other tetraspanin markers for their specific research questions, often employing multiple markers for comprehensive analysis of tetraspanin biology .