Exosome Enrichment and Tetherin Detection

Western blotting for tetherin and for CD63 was performed in non-reducing conditions and tetherin blots using a sample buffer without bromophenol blue (10% SDS, 15% glycerol, 0.2 M Tris-HCl pH 6.8) as previously described (Giese and Marsh, 2014 (link)). Other Western blots were performed according to standard protocols. Signals were quantified using ImageJ. Exosome-enriched preparations were generated from cell culture supernatants as previously described (Théry et al., 2006 (link)). Cells were grown in equal numbers on 245 mm x 245 mm dishes in 60 ml of media. Supernatants were collected and cells removed by a low speed spin (300xg, 10 min). Pellets were discarded and supernatants were then spun to remove large cell debris (2000xg, 10 min). Again pellets were discarded and supernatants were ultracentrifuged (Type 70 Ti Rotor, Beckman Coulter) to remove smaller cell debris (10,000xg, 30 min). Pellets were retained for controls and supernatants were again collected and ultracentrifuged (Type 70 Ti Rotor, Beckman Coulter) at 100,000xg for 70 min. Pellets were washed in PBS and repelleted at 100,000xg for 70 min (Type 70 Ti Rotor, Beckman Coulter) to give exosome-enriched fractions. Exosome-enriched pellets were resuspended in 100 μl sample buffer and equal volumes loaded for Western blotting. Quantification of Western blotting was performed using ImageJ.