Background: The extracellular vesicle (EV) research field has dramatically increased in the last five years. Using a high-speed flow cytometric sorter, EVs may be isolated at high rates such that researchers can differentially separate, isolate and characterize the EVs for downstream analysis. EVs contaminated with proteins, dye or antibody aggregates of the same size, but different mass, can be characterized based on these physical properties in the analytical ultra-centrifuge. This presentation focuses on characterizing the EVs on an XLA/I AUC and dynamic light scatter isolated by flow cytometric sorting. Methods: Extracellular vesicles were isolated from a HeLa cell line using serial ultracentrifugation. HeLa EVs were measured for total protein content and confirmed using CD63 Dyna-beads. EVs were stained with PKH26 and sorted on the MoFlo Astrios EQ using side scatter and fluorescence. Instrument performance was tested with polystyrene latex beads ranging from 22- 104 nm, and 100 nm liposomes for minimum detection limits. Absorbance and interference were used on AUC to measure the EV sedimentation. DelsaMax (DM) Core, dynamic light scatter (DLS) instruments, were used to measure the particle sizes of EV samples. Results: The Astrios EQ was able to distinguish and sort the EVs with some overlap on SSC noise. 100 nm liposomes and 81 nm were visible above noise. EVs stained with PKH-26 and sorted on SSC and fluorescence with populations above and in the noise. Sorted fractions were analyzed on the AUC and DM for unstained, stained and aggregate population distributions. AUC indicated EV populations with varied sizes and dye aggregates and EV size and distribution was measured by the DM with DLS. Conclusions: Sorting stained EV populations on a high speed sorter provides for their visualization, separation and characterization. The AUC effectively separated particles on their sedimentation velocity and clarified issues with dye aggregation vs EV staining. Addditionally, the DelsaMax allowed for quick analysis of post-sorted populations. The Astrios EQ was able to sort EVs and AUC provided additional analysis for exosome purity.Learning Objectives:
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