Abstract

Next generation nanoFACS with High Resolution Imaging and Custom 50um Nozzle

Terry Morgan, Professor, Oregon Health and Science University

More efficient approaches to nanoscale flow sorting (nanoFACS) are required for sufficient EV yield per volume for validation studies and more precise characterization of EV contents. NanoFACS yields approximately 1EV/1nl droplet using a 70um nozzle and 70 PSI at 75 KHz. This yeilds 106 sorted EVs/ml in one hour. RNA and protein yields are sufficient for targeted ELISA and qRT-PCR, but insufficient for –omics and bioassays. Concentrating sorted EVs by ultracentifugation, or cellulose-based 10kD filters, loses >90% of events into charged matrix. Our objective was to test whether a narrower custom nozzle at lower PSI and higher kHz could decrease nanoFACs droplet size to increase sorting concentration. We used a FACSAria Fusion customized with ZenPure PES 0.1um filter, SP-SCC small-particle analyzer module (BD Biosciences), and custom made 50um nozzle (BD). Machine settings were standardized using fluorescently labeled polystyrene beads. We imaged, counted, and sorted 40nm- and 200nm- beads, 120nm inactive murine leukemia virus +/- surface GFP (ViroFlow), FITC-conjugated 180nm liposomes [BD], and Muc5ac/CD63 positive EVs. The high resolution SP-SCC custom module provided a 2-fold increase in resolution below 100nm. We observed a 5-fold enrichment of nanoFACS sorted events with the 50um nozzle at 50 PSI at 85 kHz.