Extracellular vesicle analyses by nanoFACS

FARE Award Winner

Authors

• A Morales-Kastresana
• LM Jenkins
• K McKinnon
• TA Musich
• T Demberg
• M Terabe
• JA Berzofsky
• JC Jones

Abstract

Extracellular vesicles (EV) are heterogeneous populations of nano-sized (50-150nm) vesicles with important regulatory roles in immunity and cancer. The source and characteristics of EVs determine distinct functional roles, suggesting the existence of different EV subsets. NanoFACS method allows the analyses and sorting of single EVs according to their phenotypical charachteristics. In this study, EVs were isolated from tumor cell cultures by differential ultracentrifugation, and quantified and sized by Nanoparticle Tracking Analyses (NTA, Nanosight). NanoFACS was performed with an AstriosEQ flow cytometer, triggered with a high sensitivity SSC detector for EV analyses based on light scattering, in addition to standard FSC and FL parameters. To assess the efficiency in sorting EVs based on fluorescense, we tested several lipidic (PKH26, CM-Dil) and protein-binding (CFSE, CellTrace Violet, DDAO). Protein-binding dyes resulted in efficient EV staining without aggregate formation, particularly in the case of CFSE. For EV subset specific marker selection, we performed mass spectrometry analyses on EVs isolated from irradiated and non-irradiated 4T1 tumor cells. We found that among 160 proteins uniquely identified in EVs from irradiated cells, six were surface proteins highly overrepresented (Mcam, Plexin A1, Stomatin, Icam-1, Neuropilin-1 and Ly75). We selected these six proteins as candidates of radiation-specific markers for further study of EV subsets by nanoFACS. To leverage the tremendous potential of EVs as biomarkers and regulators of disease, we need ways to identify relevant EV subsets. We have developed a method for efficient EV staining and identified putative surface markers for further EV subset analyses by nanoFACS.

Scientific Focus Area: Research Support Services

This page was last updated on Friday, March 26, 2021