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Dr. Sascha Raschke
Particle Metrix GmbH, Inning, Germany

Introduction

The characterization of extracellular vesicles (EVs) has moved beyond simple bulk quantification toward the necessity of identifying specific subpopulations. According to the MISEV guidelines [1], EVs are suggested to be characterized by the presence of transmembrane proteins, with the tetraspanins CD9, CD63, and CD81 serving as the primary markers of identity.

While standard Nanoparticle Tracking Analysis (NTA) provides total particle concentration through light scattering, Fluorescence-NTA (F-NTA), particularly using the ZetaView® (Particle Metrix), allows for highly selective analysis of these specific markers [2]. Furthermore, the zeta potential (ZP) is a key physico-chemical parameter that indicates the surface charge, colloidal stability, and potential biological interactions of these vesicles [3, 4]. In addition, the use of antibody–fluorophore labeling introduces a critical but often overlooked challenge; depending on the labeling strategy, the binding of antibody–fluorophore complexes can alter the parameters being measured, such as the ZP of EVs.

This application note provides a methodological framework for the characterization of EV. subpopulations using the ZetaView® platform, focusing in particular on the influence of different antibody dyes and membrane stains on ZP measurements. By evaluating both, specific immunomarkers from the Particle Metrix Tetraspanin Detection Kits (488, 520 and 640), and generic membrane dyes (CellMask™), we demonstrate that total particle counts in scatter mode remained stable, while F-NTA revealed a heterogeneous tetraspanin distribution dominated by CD9. Notably, concentration measurements within each respective subpopulation (CD9, CD63, CD81) and PAN remained remarkably stable regardless of the fluorophore used. However, immunolabeling induced significant ZP shift toward positive values, particularly with 488 dyes while 640nm fluorophores demonstrated superior preservation of the native charge across all markers. CellMask™ membrane dyes on the other hand, efficiently detected 70–75% of the total scatter population while maintaining near-native ZP values demonstrating that lipophilic membrane staining causes minimal charge masking compared to protein-specific immunolabeling of EV subpopulations.

This case study aims to raise scientific awareness and highlights the importance of carefully evaluating experimental conditions when measuring ZP in specific EV (sub-) populations.

 

Materials and Methods

Sample Preparation and Labeling Strategy

Extracellular vesicles derived from the HCT 116 cell line were characterized using a panel of antibodies originated from the Particle Metrix Tetraspanin Detection Kits (488-, 520- and 640- conjugation, corresponding to the Particle Metrix article nos. #700384, #700385 and #700386). In addition, unstained EVs were employed to establish the native baseline and to detect potential shifts in ZP resulting from antibody binding. To detect any putative aggregation or particle formation among the antibodies, conjugated antibodies without EVs were measured as an internal control. Since no antibody aggregates or clusters could be detected the data are not shown. Individual CD9, CD63 and CD81 antibody labeling as well as PAN-staining were performed in a reaction volume of 20 µl. 2µl (≙ 1.3x10⁸ particles) HCT 116 EVs were incubated with 1µl of the respective antibodies (pre-diluted 1:10 in 10% PBS buffer) and 17µl 10% PBS. In addition, generic membrane labeling was performed in a reaction volume of 20µl as well. 2µl (≙ 1.3x10⁸ particles) HCT116 EVs were incubated with 1µl of CellMaskTM Green (CMG), CellMaskTM Orange (CMO) or CellMaskTM Deep Red (CMDR) (pre-diluted 1:10, 1:1.000 and 1:1.000 in 10% PBS buffer) and 17µl 10% PBS. Following the incubation period of one hour at room temperature in the dark, each reaction mixture was supplemented with 980 µl of PBS (10%) to achieve a final measurement volume of 1.000µl per sample.

Nanoparticle Tracking Analysis

Nanoparticle Tracking Analysis (NTA) was performed using a ZetaView® PMX x35 QUATT instrument (Particle Metrix, Germany) equipped with ZetaSphere software version 1.1. This device represents an upgraded version of a ZetaView® x30 model and is technically equivalent to the current ZetaView® x40 Evolution series. Prior to measurements, the scatter channel and all relevant fluorescence channels (488/500, 520/550, and 640/660) were set up by the automated set-up procedure provided by the ZetaSphere software, ensuring sharp focus across all 11 measurement positions in every available channel.

For each sample, concentration and ZP were recorded. To minimize potential photobleaching, a "fluorescence-first" strategy was applied: samples were initially analyzed in the fluorescence channel corresponding to the respective fluorophore, followed by scatter mode analysis. Additionally, the system was configured to automatically move a fresh sub-volume of the injected sample into the field of view before the start of each measurement to further reduce the detection of bleached particles. For statistical robustness, the measurements were performed in triplicates. While the EV-measuring configurations pre-installed in the ZetaSphere provide a reliable baseline, fine-tuning of the camera parameters remains essential to account for the specific physical and optical properties of the labeled EVs. The specific acquisition settings are summarized in Table 1.

Table 1: NTA-acquisition settings for scatter and fluorescence mode.

 

Results

Scatter Baseline, Concentration and Marker Expression

Across all samples, the total particle count observed in scatter mode remained stable at approximately 6.0 to 6.5×1010 particles/mL (Fig.1 grey bars). However, the F-NTA concentration data showed significant heterogeneity in the frequency of the distinct tetraspanin markers regardless of which dye (488, 520 or 640) was used (Fig.1 colored bars): CD9-positive vesicles appear to be the most abundant single-marker population across all single labelings with concentrations reaching between 3.4x10¹⁰ to 3.6x10¹⁰ particles per ml. CD81 indicated intermediate abundance, showing lower detection rates (5.7x10⁹ to 1.1x10¹⁰ particles per ml) than CD9 but slightly higher than CD63 (4.7x10⁹ to 5.3x10⁹ particles per ml). The PAN-mix (CD9/63/81) provided stable overall fluorescence detection, slightly lower but similar to the CD9 fraction, showing a concentration range of around 3x10¹⁰ particles/mL, apparently capturing a broad representation of the total EV population. It is important to note that when considering the individual CD9, CD63 and CD81 subpopulations, the measured concentrations were approximately the same, which suggests dye-independent homogeneous labeling.

Figure 1: Concentration of particles positive for the individual markers CD9, CD63, CD81 and PAN, detected via F-NTA for the wavelengths λ=488nm, λ=520nm, λ=640nm. Within each subpopulation, the concentration of labeled particles is approximately the same, regardless of the dye used.

Zeta Potential and Fluorophore Effects

The native Zeta Potential of unstained EVs, as well as that of labeled ones detected in the scatter mode, was measured at −28.5 mV to −30.5 mV, indicating a highly negative, stable surface charge typical for mammalian EVs (see Fig. 2 grey bars). This indicates that the presence of antibodies in the buffer does not fundamentally change the charge of the unlabeled background population, providing a reliable biological zero point.

However, staining of EVs with all tested antibodies resulted in a significant shift in ZP toward a less negative surface charge (Fig. 2 colored bars). The magnitude of this shift from the native ZP of around −29 mV toward less negative values depended on both the marker choice and the fluorophore. Across all markers, the 488 dye induced the strongest shifts in ZP. The most pronounced effect was observed for CD63 labeled with the 488 dye, reaching a ZP of −5.3 mV. In contrast, the 640 dye showed the best preservation of the native surface charge, with 640-conjugated CD9, CD81, and PAN samples exhibiting ZP values below −20 mV. 

Notably, the best preservation of the native ZP was observed for PAN and CD9 staining, which also showed the highest particle concentrations among the analyzed subpopulations.

Figure 2: Zeta potential of particles positive for the individual markers CD9, CD63, CD81 and PAN, detected via F-NTA. Within each subpopulation, the 488 dye produces the largest ZP shift, while the 640 dye best preserves the native state of the ZP.

Impact of Unconjugated Antibody Binding

A critical control experiment showed that the binding of CD9, CD63 and CD81 antibodies without fluorophores (unconjugated) caused no significant change in concentration or ZP. This result proves that the physical integrity and surface charge of the EVs are not compromised by the antibody- protein interaction alone. ZP shifts mentioned above are therefore considered to be a direct effect of the labeling of fluorophore conjugated antibodies.

Figure 3: Concentration (A) and ZP (B) of particles labeled with unconjugated CD9, CD63, CD81 antibodies, detected in scatter mode. No effect on the concentration or surface charge was observed when unconjugated antibodies were used alone.

Detection Efficiency and Charge Stability of Membrane Staining

Labeling with CellMaskTM dyes demonstrated excellent performance for total population detection. Membrane staining detected approximately 70–75% of the scatter population across all wavelengths (Figure 4 colored bars). Remarkably, the ZP remained nearly native. While the unstained control was at −26.5 mV, the stained particles showed values from −25.2 to −27.6 mV indicating minimal charge masking.

Figure 4: Concentration (A) and ZP (B) of particles labeled with CMG, CMO and CMDR, detected in scatter and fluorescence mode. No significant change in ZP could be detected.

 

Conclusion

This study demonstrates that the ZetaView® platform provides a robust dual-mode approach for characterizing both the total EV population and specific tetraspanin-positive subpopulations. While scatter mode analysis confirms a stable baseline concentration, F-NTA reveals significant heterogeneity in marker expression in this HCT116 EV model. Crucially, concentration measurements within each subpopulation (CD9, CD63, CD81) and PAN remained remarkably stable, regardless of the fluorophore wavelength used, indicating consistent and reproducible labeling efficiency across the spectrum.

Despite this stability in concentration, the analysis reveals that ZP is significantly influenced by both the antibody (marker choice) and the conjugated fluorophore. While native EVs maintain a stable surface charge, specific immunolabeling induces a shift toward less negative ZP. This effect is most pronounced with 488nm fluorophores. In contrast, CellMask™ membrane dyes maintained a constant, near-native ZP, providing excellent detection efficiency without significant charge masking.

These results demonstrate that the chemical integration of CellMask™ dyes into the lipid bilayer preserves the native biophysical identity of the EVs, making them a robust tool for ZP analysis of all existent membranous particles in the sample including EVs. Conversely, the specific nature of immunolabeling requires careful consideration of the signal-to-charge ratio. For protein-specific analysis utilizing the most sensitive detection channels, it is important to ensure that the measured ZP best reflects the vesicle's biology. This study also demonstrates that the ZP in F-NTA is a dynamic indicator of antibody-target interaction.

Since Zeta Potential is inherently dependent on a multitude of variables, including buffer composition, pH levels, salt conditions and specific antibody characteristics, these findings should be regarded as a contextual case study rather than a universal standard. This study aims to strengthen scientific awareness and encourage rigorous evaluation of experimental parameters when performing subpopulation-specific ZP measurements on extracellular vesicles.

 

References

1. Théry, C., et al. (2018).: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). Journal of Extracellular Vesicles, 7(1), 1535750.

2. Kormutak, R., et al. (2019).: Characterization of extracellular vesicles by nanoparticle tracking analysis: a comparison of scatter and fluorescence mode. Journal of Extracellular Vesicles, 8(1), 1664510.

3. Midekessa et al., (2020).: Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability. ACS Omega 30;5(27):16701-16710.

4. Gardiner, C., et al. (2013).: Techniques used for the isolation and characterization of extracellular vesicles. Journal of Extracellular Vesicles, 2(1), 19671.