Profiling Extracellular Vesicle-Associated FHR-1

Vesicles isolated by precipitation from human serum were incubated with monoclonal anti-human FHR-1^{10 (link)} antibody conjugated with Alexa Fluor 647 (1:200) to detect FHR-1. FHR-1-transporting EVs were captured using beads coated with monoclonal FHR-1 antibody and were stained with Alexa Fluor 647-labeled anti-human CD9 antibody (1:100; Novus Biological, cat no. NB500-327). Samples were transferred into 35 mm culture dishes with a glass-bottom (Greiner). To detect nucleic acids in vesicles, the vesicles were incubated with 5 µM SYTOX orange (Thermo Fisher Scientific). Pictures were taken with LSM and analyzed using ZEN 2011 software.
PLA assays were performed to evaluate the co-localization of the vesicle markers CD9 and FHR-1. Vesicles isolated by precipitation from human serum were seeded onto 6.7 mm diagnostic slides coated with poly-l-lysine. The vesicles were fixed with 4% formaldehyde, blocked with Duolink blocking solution (Sigma-Aldrich), and incubated with mouse antibody to human FHR-1^{13 (link)} (1:200) and rabbit antibody to human CD9 (1:200; Abcam, cat no. ab92726). PLA assays were performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, cat no. DUO92101) according to the manufacturer’s instructions. Images were captured with LSM 710 fitted with ZEN 2011 software.