TABLE 2.
Methods of EV analysis and their limitations.
| Method | Strengths | Pitfalls/Limitations | References |
|---|---|---|---|
| Flow cytometry | • Fast method with single particle resolution and excellent statistics | • smaller EVs are not detectable (lower limit: about 100–300 nm) | (Ayers et al., 2011; van der Pol et al., 2014; Fendl et al., 2016; Théry et al., 2018; George et al., 2021) |
| • Fluorescent antibodies or lipophilic dyes can be applied for specific detection | • Equipment not available in all labs | ||
| • Combination with degradation approaches or antibodies binding to extravesicular domains allows detection of inside-out EVs | • Results may vary between labs (dependent on instrument settings) | ||
| • Combined with flow sorting it can be used to purify subpopulations for further differential analysis | • Proper gating strategy is important to analyze the correct set of particles (raw data and gating are often not shown in publications for all results) | ||
| Nanoparticle tracking analysis (NTA) | • Precise determination of size range and concentration is possible | • Light-scattering mode does not discriminate between membranous vesicles and protein aggregates or lipoproteins | (Saveyn et al., 2010; van der Pol et al., 2014; Holnthoner et al., 2017; Vestad et al., 2017; Théry et al., 2018; Bachurski et al., 2019; George et al., 2021) |
| • Fluorescence mode particle tracking allows more specific detection of EVs | |||
| Biochemical analyses (SDS-PAGE, Western Blots, etc.) | • Allow more specific analysis of EV components | • Require a correct purification of EVs | (Thomas et al., 2009; Iwai et al., 2016; Fendl et al., 2018; Théry et al., 2018; Lee et al., 2019; Tripisciano et al., 2020; Zhang et al., 2020) |
| • Can be used to determine contaminants and the purity of EV preparation | |||
| Next Generation sequencing approaches or proteomics | • Unbiased and integrative analysis of EV constituents and cargo | • Expensive and requires access to core facilities of sequencing or proteomics | (Laroche et al., 2017; Théry et al., 2018; Veziroglu and Mias, 2020; Xu et al., 2020; Hildebrandt et al., 2021) |
| • Can be used for differential analysis (e.g., disease versus control) | • bulk sequencing or proteomics does not allow to determine subpopulations of EVs (has no single particle resolution) | ||
| Electron microscopy | • Provides high resolution images of EVs and size information | • low-throughput and tedious method | (Arraud et al., 2014; van der Pol et al., 2014; Cizmar and Yuana, 2017; Théry et al., 2018) |
| • can differentiate between EVs and lipoprotein particles or protein aggregates | • equipment often not available | ||
| • mostly unspecific as immuno-EM is difficult and sometimes impossible |