Nanoparticle Isolation via Nano Scale Sorting: Small Footprint, Small Particles, and Big Ideas

In recent years extracellular vesicles (EVs) have become one of the most studied entities due to their ability to regulate gene expression and alter the function of various cell types. There has been significant interest in the cargo and the cells of origin due to this effect. Nano-flow cytometry is one of the techniques employed in these characterization efforts. ​ With hardware advancements and the increase in use of flow cytometry to analyze EV populations, there have been efforts to standardize nano-flow data for ease of comparison and collaboration. While these efforts have made progress for nano-flow analysis, nano-sorting is still a complex technique that is not commonly used. It has been previously demonstrated, on a MoFlo Astrios (Beckman Coulter, Brea, CA), that nanoscale vesicles could be sorted with efficiencies ranging from 25 to 45%. In an effort to increase this efficiency, we employed the use of the CytoFLEX SRT (Beckman Coulter, Brea, CA) to enrich EV populations and enhance miRNA detection via molecular beacons. ​

 

Due to the complimentary nature of the CytoFLEX SRT and the CytoFLEX S, both use APD detectors, WDM, and VSSC detection, it was deemed as a suitable instrument for sorting nanometer sized particles. In addition, the automated sort setup and minimal instrument modifications necessary to perform nano-flow cytometry made the instrument an attractive option for standardized and reproducible experiments. Therefore, multiple nanometer beads and specimens were analyzed and sorted.​

 

Previously, Pereira et al described successful analysis of the miRNA content of red blood cell derived EVs (RBCEVs) using MBs and nano-flow cytometry. Therefore, in order to validate the plausibility of the CytoFLEX SRT as a small particle sorter, miR451a specific molecular beacons (MBs) were used and FL positive high and low populations were sorted. In addition, whole plasma was labeled with a fluorogenic membrane dye to sort EV populations from total plasma, removing all contaminants without enrichment.

 

Learning Objectives