Table 3.
Main techniques for EV analysis.
| Methods for quantifying EVs | |||
|---|---|---|---|
| Method | Description | Advantages | Disadvantages |
| Nanoparticle tracking (NTA) | Tracks the Brownian motion of the particles in scattering or in fluorescence mode and and by measuring the scattering intensity of single particles infers their size | Accurate for both monodisperse and polydisperse; calibration particle standards | Size >70 nm; requires specific instruments (Nanosight and Zetaview) |
| Dynamic light | Analysis the fluctuations of scattering intensity of particles in Brownian motion | Accurate for monodisperse samples; lower size (< 30 nm) | Large particles can compromise the results, inaccurate for polydisperse samples; specific instrument required (NanoZS and Nanoflex); requires a high concentration of monodisperse particles to be detected, which is not convenient for low yields of collected EVs |
| Resistive pulse sensing | Measures the change in conductance across a sensing pore upon passage of a particle | Surface charge | For unknown size distribution, insufficient for detection of all particles, size >70 nm. Requires specific instruments : qNano |
| Flow cytometry | Measures scattering or fluorescence intensity of particles illuminated by a laser | Low particle concentration (106 particles ml−1) | Size >200 nm. For EXOs not absolute size measurement. A flow cytometer required |
| Electron microscopy: cryo-EM (cryoelectron microscopy)and TEM (transmission electron microscopy) | Utilizes electrons instead of photons to create an image with a resolution down to the nanometer | TEM/cryo-EM: direct visualization and observation of EVs, EV structure/morphology; cryo-EM: preserves membranes in native state | TEM: fixation induces shrinking of EV structure, equipment: electron microscope, cost |
| Immunoblotting (IB) | IB is based on the detection and relative quantification of EVs by using specific antibody against characteristic markers such as CD9, CD63, CD81, TSG101, Alix, actin, tubulin, flotillin-1, HSC70/HSP73, HSP70/HSP72, and MHC molecules | In combination with other techniques IB is largely used to characterize and assess the degree of purity of EV preparations: the absence of cell-derived organelle markers such as calreticulin is often used to assess the purity of an EV preparation | IB cannot be used to quantify EVs and the enrichment of these proteins in the EV fraction does not guarantee the absence of contaminants |