(a) Exosome purification schematic. (b–e) Representative electron micrographs of negative stained samples from the 100,000 ×g pellet fraction (b,d) and post-flotation fractions (c,e) at either 9300X (a,b) or 1900X (c,e) magnification. Open arrows indicate large (>200 nm) vesicle contaminants and closed arrows indicate protein aggregates. (f) CD63-luciferase activity in purified exosomes after treatment with 1% Triton X-100 (TX-100) and/or 100 µg/ml trypsin for 30 min at 4°C. Error bars represent standard deviations from 3 independent samples. (g) Specific activity of CD63-luciferase (RLU/µg of total protein) at each stage of purification (green: 100,000 ×g pellet, purple: post-flotation, red: post-immunoisolation a-CD63 beads). (h) Total RNA recovered from conditioned medium after immuno-isolation with a-CD63 or an IgG control. B – bound to beads, FT – flow-through not bound to beads. Error bars represent standard deviations from 3 separate purifications (biological replicates).