Extracellular Vesicle Measurements with Nanoparticle Tracking Analysis: A Different Appreciation of Up and Down Secretion

Abstract

As is the case with most eucaryotic cells, cancer cells are able to secrete extracellular vesicles (EVs) as a communication means towards their environment and surrounding cells. EVs are represented by microvesicles and smaller vesicles called exosomes, which are known for their involvement in cancer aggressiveness. The release of such EVs requires the intervention of trafficking-associated proteins, mostly represented by the RAB-GTPases family. In particular, RAB27A is known for its role in addressing EVs-to-be secreted towards the the plasma membrane. In this study, shRNAs targeting RAB27A were used in colorectal (CRC) and glioblastoma (GB) cell lines in order to alter EVs secretion. To study and monitor EVs secretion in cell lines’ supernatants, nanoparticle tracking analysis (NTA) was used through the NanoSight NS300 device. Since it appeared that NanoSight failed to detect the decrease in the EVs secretion, we performed another approach to drop EVs secretion (RAB27A-siRNA, indomethacin, Nexihnib20). Similar results were obtained i.e., no variation in EVs concentration. Conversely, NTA allowed us to monitor EVs up-secretion following rotenone treatment or hypoxia conditions. Therefore, our data seemed to point out the insufficiency of using only this technique for the assessment of EVs secretion decrease.

An optimized workflow for analyzing extracellular vesicles as biomarkers in liver diseases

“…NTA is a technique based on light-scattering, which enables the rapid sizing and enumeration of nanosized particles in solution [17]. Since its introduction, NTA has become a standard procedure to reproducibility measure EVs size and concentration in their native conditions [18, 19]. Importantly, precision and reproducibility of NTA measurements was carried out by using standardized procedure (Supplementary Figure 3, Supplementary Figure 4, and Supplementary Table 2).…”

Comparison of the Exosome Profile in Ankylosing Spondylitis and Healthy Controls

“…However, none of these methods alone is sufficient for isolation and downstream analysis, as EVexo has a similar size, molecular content, and origin to many non-EV structures (11,12). Therefore, in the literature, it is recommended to combine more than one method to isolate and characterize EVexo (11)(12)(13)(14)(15)(16)(17)(18). We also used different methods (SEC, FC, BCA, and ELISA) that support and complement each other, based on the research question and downstream applications, to validate the results and design a validated algorithm for downstream studies.…”

Engineered bone marrow mesenchymal stem cell-derived exosomes loaded with miR302 through the cardiomyocyte specific peptide can reduce myocardial ischemia and reperfusion (I/R) injury

“…Later on, the size distribution of exosomes was identified using Nanoparticle tracking analysis (NTA; Nano-Sight LM10; Germany) with particular parameters that were set based on specifications of the manufacturer. In brief, we carried out the exosomal quantification using method described earlier [ 21 ]. Through the above-mentioned NTA, we captured and analyzed the exosomes with built-in Nano-Sight software.…”