Extracellular vesicles (EVs) are minuscule, membrane-bound structures that cells release into their environment. These vesicles act as cellular couriers, transporting proteins, DNA, and RNA molecules to other cells. Over the last decade, EVs have garnered significant attention due to their potential as natural carriers for complex cellular components and their involvement in various physiological and pathological processes. 

Why EV Labeling Matters?

A multitude of in vitro and in vivo techniques are employed to delve into the biological functions of EVs. These include flow cytometry, confocal microscopy, and in vivo fluorescence detection methods. These techniques often rely on labelling EVs to facilitate their detection and analysis. Labelling strategies encompass optical (fluorescence, bioluminescence), nuclear, and magnetic resonance imaging (MRI) tracers, as illustrated in Figure 1. These advanced methods enable comprehensive insights into EV functions, interactions, and roles in physiological and pathological processes. 

Figure 1: Strategies for labelling of exogenous extracellular vesicles. 

Credit: Boudna A., Campos AD., et. al., https://doi.org/10.1186/s12964-024-01548-3 

Reproduced under the Creative Commons license 

Fluorescent Dyes for EV Staining: Current Approaches and Limitations

Detecting and characterising small EVs poses a significant challenge in the field. Flow cytometry, however, is frequently utilised for its ability to perform rapid, high-throughput, multiparameter analysis. Various fluorescent probes are commonly employed to detect small EVs using flow cytometry, including: 

1. Antibody-Based EV Stains

These dyes exploit specific interactions with surface markers on small EVs, allowing for precise and targeted detection. These dyes are conjugated to antibodies that recognise and bind to distinct proteins on the EV membrane, facilitating their identification and characterisation. Notable surface markers include the tetraspanins CD9, CD63, and CD81. These proteins are highly enriched on the surface of EVs and are commonly used as markers for their detection. Antibody-based dyes targeting these tetraspanins provide specificity and sensitivity enabling the detailed analysis of EV populations, including their origin, composition, and potential functions. This targeted approach enhances the accuracy of flow cytometry in EV research, providing valuable insights into their roles in various physiological and pathological processes. 

2. Membrane-Permeable EV Stains

Membrane-permeable dyes, such as carboxyfluorescein diacetate succinimidyl ester (CFSE), play a critical role in labelling and tracking EVs. CFSE is a lipophilic fluorescent dye that passively diffuses into cells and becomes highly fluorescent when intracellular esterases cleave its acetate groups. This reaction results in the dye covalently binding to amine groups in intracellular proteins, producing a stable and intense fluorescence signal. However, antigen expression and intracellular protein content vary depending on the cellular origin, leading to heterogeneity within a small EV population. Consequently, CFSE labelling may not be optimal for comparison across different samples. Additionally, the dye can leak from labelled cells, staining the surrounding environment and creating background noise that can obscure EV signals. 

3. Lipophilic EV Stains

A variety of commercial lipid dyes are available, including PKH dyes and carbocyanine dyes (such as DiD, DiR, DiI). PKH dyes are highly fluorescent cell linkers that integrate into the cell membrane’s lipid bilayer. Common examples include PKH26 (red) and PKH67 (green). Although PKH dyes are widely used for labeling small EVs, there is growing consensus that they are unsuitable because they cause significant aggregation and alter vesicle size, and their uptake by cells produces false positives that compromise the reliability of experimental results. Studies have shown that PKH dyes can bind non-specifically to other cellular components or form free dye aggregates, creating background noise and complicating the identification of labelled EVs. PKH dyes might also affect membrane–membrane fusion, fluidity of membrane proteins, membrane stiffness and EV size.  

Carbocyanine dyes, such as DiO and DiI, are another class of lipophilic dyes used for EV labelling. These dyes are known for their bright fluorescence and compatibility with various imaging techniques. However, these dyes suffer from poor solubility, leading to the formation of aggregates and fluorescent micelles that can easily be misinterpreted as EVs. This not only reduces labeling accuracy but also interferes with flow cytometry and fluorescence nanoparticle tracking analysis (fNTA), resulting in misleading data.

While lipophilic dyes like PKH and carbocyanine dyes are valuable tools for EV labelling, their limitations necessitate careful consideration and experimental design. Researchers must account for potential non-specific binding, aggregate formation, and impacts on membrane properties to ensure accurate and reliable EV detection. Exploring alternative dyes and labelling techniques may help mitigate these issues and enhance the precision of EV studies. 

ExoBrite™: Next-Generation Dyes Optimized for EVs

Maintaining the integrity of EVs while ensuring accurate fluorescent labelling is paramount for reliable analysis. For extracellular vesicle membrane staining, modern purpose-built dyes such as Biotium’s ExoBrite™ series overcome these limitations by offering optimized chemistry specifically designed for EV research. Biotium has revolutionised EV labelling with their groundbreaking membrane dyes, offering a remarkable improvement over conventional methods. 

ExoBrite™ True EV Membrane Stains – Best Pan-EV Coverage

ExoBrite™ True EV Membrane Stain represent a breakthrough in EV labeling technology, specifically optimized for extracellular vesicles. These stains provide higher, near-complete coverage of EVs within a sample, ensuring efficient labeling with minimal aggregation.

Unlike traditional dyes such as PKH, DiO, DiI, and DiD — which often form aggregates, distort vesicle size, and produce false positives — ExoBrite stains deliver bright, specific signals that can be clearly distinguished from nonspecific particles. Crucially, they are designed to address aggregation issues, allowing for clear and reproducible differentiation of EVs from background during flow cytometry detection and other analytical workflows.

Customer Testimonial – NTU Researcher
“ExoBrite stains EV membranes very effectively and provides clear visualization. It works especially well after SEC column purification, giving confidence that isolated EVs are being detected. Even with just 1 µL of stain, the signal is sharp and reliable.”

This makes ExoBrite staining highly reliable for flow cytometry, fNTA, and advanced imaging, providing reproducible, publication-quality EV analysis and advancing the precision of EV research.

ExoBrite™ EV Surface Stains

Biotium’s ExoBrite™ EV Surface Stains provide a targeted approach to labeling extracellular vesicles by binding to specific EV membrane surface. Each stain offers unique advantages depending on the biological question:

Together, these surface-target specific EV stains expand the analytical toolkit for researchers by enabling selective labeling of biologically relevant EV membrane biomarkers. By minimizing aggregation compared to conventional dyes, ExoBrite™ EV Surface Stains provide clear and distinct signals for both purified and bead-bound EVs. This advanced technology supports accurate identification and characterization of EV subpopulations, offering researchers reproducible, high-resolution data and unlocking new opportunities in EV biology and biomedical research.

ExoBrite™ STORM CTB EV Stains for Super-Resolution Imaging 

The introduction of innovative dyes like ExoBrite™ by Biotium represents a significant leap forward in EV labelling and imaging technology. By overcoming the limitations of traditional dyes such as PKH and carbocyanine, these advancements provide researchers with reliable and precise tools for studying EV biology. These groundbreaking technologies not only enhance our understanding of EVs but also hold immense promise for their applications in medical research. 

Antibody Conjugates for CD9, CD63, CD81

Biotium’s ExoBrite™ Antibody Conjugates are designed to provide researchers with highly specific tools for detecting canonical EV membrane biomarkers. These reagents target the tetraspanins CD9, CD63, and CD81, which are the most widely recognized markers for EV identification and characterization. By using optimized formulations, ExoBrite antibodies deliver brighter signals, reduced aggregation, and superior signal-to-noise, making them reliable across multiple EV analysis platforms.

These antibody-based tools ensure high specificity and minimal background, enabling accurate characterization of EV populations across flow cytometry, Western blot, and super-resolution microscopy workflows.

Best Practices for Reliable EV Labeling

To ensure reliable and publication-quality EV data, researchers should adopt rigorous controls and purification methods alongside their staining workflow:

• Use dye-only controls: Detect and account for background fluorescence from free dye aggregates.
• Apply detergent lysis controls: Confirm that fluorescence signals originate from lipid-bound EVs rather than contaminants.
• Purify EVs before staining: Biofluids such as plasma contain lipoproteins that vastly outnumber EVs and can also be stained by lipophilic dyes. Proper purification (e.g., size exclusion chromatography) before staining is essential to remove these contaminants and improve labeling specificity.
• Perform post-staining cleanup: After labeling, additional cleanup steps such as SEC columns or spin filters can be used to remove unbound dye and reduce background noise.
• Balance sensitivity with specificity: Pan-EV membrane stains offer broad coverage, while surface-targeted stains or antibody conjugates allow precise analysis of EV subpopulations.

By integrating these best practices with optimized reagents such as ExoBrite™, researchers can achieve both technical rigor and biological accuracy, ensuring confidence in downstream EV analysis.

References: 

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