Exosome Isolation by Miniaturized Size Exclusion Chromatography

Culture supernatants or freshly-thawed plasma were centrifuged at 2,000 × g for 10 min at room temperature (RT) and at 10,000 × g for 30 min at 4°C followed by filtration on 0.22 μm syringe-filters (Millipore). Pre-conditioned supernatants were concentrated from 50 to 1 mL on Vivacell 100 filter units (MWCO 100,000, Sartorius Corp, Bohemia, NY, USA). Aliquots (1 mL) of pre-conditioned plasma or concentrated supernatants were loaded on mini-SEC columns (18 (link)), and exosomes were eluted with PBS. Exosomes were collected in the void volume fraction #4 (1 mL). For some experiments, particularly for Western blots, #4 miniSEC fractions were concentrated using 100,000 MWCO Vivaspin 500 Centrifugal Concentrators (Sartorius Corp) by centrifugation at 2,000 × g for 10–15 min.