**4.3. Flow cytometry analysis of extracellular vesicles**

vesicles or any of the specific populations, like exosomes or microvesicles. To stain EVs lumen, widely used dyes such as CFSE and CalceinAM may be used. Those dyes in non-fluorescent form passively enter EVs and due to esterase activity in the EV lumen are transformed into fluorescent molecules. Gray et al. demonstrated that CalceinAM stains only intact EVs, which may be advantageous for its use in both EVs uptake studies and direct flow cytometry of EVs [52]. CFSE staining was applied to quantitatively show differential association of cancer EVs with myeloid cells depending on their differentiation into various stages of monocyte and dendritic cell development [53]. In our experiments, we have used CFSE labeling to study the dose-dependent uptake of EVs released by murine BCR-ABL-expressing cells by thymocytes (**Figure 6**). It is clearly visible that increasing amounts of fluorescently labeled EVs correlate

**Table 2.** Fluorescent dyes used to fluorescently label extracellular vesicles for analysis by flow cytometry; function,

560 (yellow) PKH26 567

633/640 (red) DiD 665

**Function Excitation laser (nm) Dye Emission (nm)**

CalceinAM 515

DiO 501

Dil 565

Lumen labeling 488 (blue) CFDA-SE (CFSE) 517

30 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Membrane labeling 488 (blue) PKH67 504

excitation, and emission spectra as well as full name are shown.

Membrane staining is also widely used to label EVs with the use of dyes such as PKH67, PKH26, DiD, Dil, and DiO. They allow a wider choice of fluorescence spectra but, as they are lipophilic, can form various aggregates and micelles. Thus, in this case, EVs need to be carefully washed to avoid adding unbound dye which may nonspecifically label cells. PKH67

**Figure 6.** CFSE labeling to study uptake of EVs. Leukemia-released EVs were labeled with CFSE and different amounts of EVs were added to thymocytes. Fluorescence of CFSE in thymocytes was detected by flow cytometry in FITC channel.

with increased fluorescence of CFSE signal detected in acceptor thymocytes.

Flow cytometry has emerged as one of the tools for analysis of extracellular vesicles and their composition. As detection limit of conventional flow cytometers is around 500 nm, bead capture of EVs has been used to facilitate, for example, analysis of proteins expressed on their surface. However, direct flow cytometry of extracellular vesicles has also been widely performed [55]. As light scatter triggering itself is not sufficient to distinguish nanoscale vesicles, fluorescence triggering has been used to directly identify EVs on flow cytometers. Commercially available flow cytometer (BD Influx) has been adapted for high-throughput analysis of fluorescently labeled vesicles. PKH67 staining of EVs allowed fluorescence-based thresholding of events, and by modifying cytometer elements, the authors were also able to observe size distribution of exosomes in the FSC channel. The described technique allowed to identify molecules expressed on the surface of EVs [56] and then apply the method to perform quantitative and qualitative analyses of dendritic cell-released EVs upon different stimulations and culture conditions [57]. Direct flow cytometry of fluorescently labeled EVs using conventional flow cytometers has been applied to study mesenchymal stromal cell markers (CD90, CD44, CD73) on EVs released by HS-5 and K562 cells as well as to analyze surface molecules expressed on microvesicles isolated from patient sera with hematological tumors (CD20, CD38, CD30) [48].

However, direct flow cytometry of EVs needs to be used cautiously, as different sample parameters and cytometer settings may affect measurements. It was demonstrated that due to high concentration of very small molecules in suspension, the measurement may not be precise, due to analysis of multiple events as one. Flow cytometry is emerging as a tool for direct analysis of EVs and their surface markers, allowing fast and precise measurements. Various aspects of EVs analysis by FACS are demonstrated in a special issue of Cytometry Part A Journal "Measurement of Extracellular Vesicles and Other Submicron Size Particles by Flow Cytometry" [58]. It is noteworthy that due to small size of EVs precise fluorescent labeling needs to be applied and conventional flow cytometer settings need to be cautiously optimized, for example, by using fluorescent beads of different sizes (e.g. 200 and 100 nm beads).
