Single-Particle and Multicolor Analysis of Extracellular Vesicles (EVs)

Written by: Yasuma Yoshizumi, Alex Rodriguez, Richard Cuthbert

Introduction

Extracellular vesicles or EVs are a general term for nanosized vesicles secreted by almost all cells that have a lipid-bilayer membrane structure. They are defined as nanosized particles that are naturally released, covered by a lipid bilayer, and lack a nucleus, and cannot self-replicate (Figure 1).

EVs mainly function as carriers that transport various biomolecules such as mRNA, miRNA, proteins, and lipids, thereby promoting intercellular communication. They are also involved in many important in vivo functions, including regulation of gene-transcription rates and induction/modulation of immune responses.

EVs can also be collected from blood and retain features from their cells of origin. These characteristics mean that EVs hold potential for use as biomarkers, making liquid biopsy and EV analysis a useful strategy in the future for clinical diagnosis.

Furthermore, in recent years, research and clinical trials on disease treatment using mesenchymal-stem-cell (MSC)-derived EVs have been examined and are attracting attention worldwide. Here, using the ZE5 Cell Analyzer, we present an example of single-particle and multicolor analysis of culture-cell-derived EVs.

Fig. 1. EV release and structure.

Materials and Methods

Sample Staining and Analysis

Cells were grown in standard cell culture conditions and EVs were isolated from culture supernatants using ultracentrifugation (Figure 2).

Fig. 2. Sample workflow.

Following centrifugation, EVs were resuspended in PBS at the concentration stated in Table 1. Incubation with dyes and reagents was performed at room temperature for 30 minutes prior to analysis at the concentrations stated in Table 2. To perform compensation and to set appropriate gating, single-stain EVs and FMO controls were used. Buffer-only, antibody/dye-only, and unstained EVs were also used to control for instrument noise and stain aggregation.

Table 1. Solutions preparation.

Table 2. Reagents used.

APC, allophycocyanin; PE, phycoerythrin.

Analysis was performed using a 5-laser ZE5 Cell Analyzer equipped with a small particle detector (SPD) (12004279) according to the conditions specified in Table 3. Following data acquisition, data analysis was performed using Everest Software and FCS Express (Dotmatics).

Table 3. Instrument run conditions.

Results

Confirmation of EV Detection in Cell Culture Supernatants

Flow cytometry confirmed the presence of EVs from cell culture supernatants. Blank control PBS showed only low scatter, fluorescence-negative signal. Whereas cell culture supernatants showed a clear population of EV particles stained with FI7510. Comparison of forward scatter intensities to size standard beads showed that the majority of EVs were between 240 and 500 nm in size (Figure 3).

Fig. 3. EV detection in cell culture supernatants. Representative scatter plots showing 405 nm forward scatter against FI7510 fluorescence in PBS background (left panel). FI7510-stained cell culture supernatant derived EVs (center panel) and size comparator beads (right panel). FITC, fluorescein isothiocyanate; FSC, forward scatter; EV, extracellular vesicle.

Confirmation of Protein Expression in Culture-Derived EVs

We next determined if EVs expressed proteins of interest. Following staining with FI7510 and incubation with antibodies to proteins of interest, Protein X and Protein Y. In comparison to antibody-only controls, EVs showed clear expression of Protein X and Y. Dual staining showed 27.7% of EVs were double negative for both proteins, 15.3% of EVs expressed Protein X only, 6.2% of EVs expressed Protein Y only, and 50.8% were double positive for both proteins (Figure 4).

Fig. 4. Expression of Protein X and Y on tissue culture-derived EVs. Flow cytometry dot plots showing expression of Protein X and Y on the surface of cell culture supernatant-derived EVs. Cell-derived EVs were compared to antibody controls (antibody only, no EVs) with regard to their expression of markers X and Y individually (A). Following positive gating for FI7510 fluorescence, single staining of cell culture supernatant-derived EVs stained with marker X or marker Y or both was compared to an unstained control EV sample (B). APC, allophycocyanin; FSC, forward scatter; EV, extracellular vesicle; PE, phycoerythrin.

Conclusions

In this study, membrane staining and multiplex antibody staining were used to detect culture-cell-supernatant-derived EVs at the single-particle level using flow cytometry, while simultaneously analyzing their membrane-surface antigens. We demonstrated positive expression of the proteins of interest and quantified their expression in cell culture supernatant-derived EVs.

The ZE5 Cell Analyzer combines high-sensitivity, high-resolution measurement with multiparameter analysis, robust software- and hardware-based noise reduction, and high-speed acquisition of up to 100,000 events/sec. These capabilities enable single-EV analysis, which has previously been challenging, and support applications such as clinical-sample EV population analysis, therapeutic EV formulation quality assessment, and EV formulation storage-stability studies. The loader’s 4–37??? temperature-control function further supports EV formulation development workflows.

In summary, the ZE5 Cell Analyzer offers a powerful platform for measuring and analyzing both cell-sized particles and smaller EV-like particles, including at the single-particle level.

Would you like to speak to an expert about small particle analysis?