Plasma EV Profiling via Flow Cytometry

EVs from 20 µl plasma were utilized for flow cytometry-based profiling. EV pellets were resuspended in double-filtered PBS (df-PBS) by 100 nm filters (MilliporeSigma) and stained with fluorescence-conjugated antibodies against human fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG) (Novus Biologicals). The high-resolution Sony MA 900 Multi-Application Sorter (Sony Biotechnology) was configured to ensure acquisition events of df-PBS <10 events/second; relative size distribution of EVs were estimated using reference beads with mean size 100, 1000 and 6000 nm (Bangs Laboratories) (6 (link)). The fluorescence background was determined using unstained and antibody-stained EVs and UltraComp™ eBeads Plus (ThermoFisher Scientific). The percentages (%) of plasma EVs carrying each surface marker were determined using flow cytometry; flow cytometric data analysis was performed using FCS Express 5 software (De Novo Software).