Exosome Immunoassay Protocol

Pre-packed Exo-Spin size exclusion columns (Cell Guidance Systems) were washed with 6 mM EDTA in PBS, then 2 ml of PD effluent samples were added, and 15 × 1 ml aliquots were collected. RNA was extracted from 500 μl of each aliquot; the remainder was used for plate-based immuno-assays. Column fractions were bound overnight to protein-binding ELISA plates (Greiner Bio-One) and blocked with 1% (w/v) BSA in PBS. Bound material was detected with primary antibodies against CD9 (clone 209306; R&D Systems), CD15 (clone W6D3; BD Biosciences) or human serum albumin (clone 188835; R&D Systems), and developed using goat anti-mouse-biotinylated antibodies (Perkin Elmer) and europium-conjugated streptavidin (Perkin Elmer). Signals were measured by time-resolved fluorometry, using a Pherastar FS plate reader (BMG Labtech)^{32 (link)}.