Type 70 ti rotor

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.

Super Dome cells were grown to confluency in 30× T175 flasks. Each flask of cells was incubated with 5 ml cell culture medium containing trypsin-activated Etx (25 µg ml⁻¹; 50× the average minimum dose required to lyse 100% cells) at 37 °C for 1 h to allow binding and pore formation. Lysed cells were centrifuged at 1500 × g for 15 min at 4 °C to pellet cellular debris and unbound Etx was removed by ultracentrifugation at 100,000 × g for 1 h at 4 °C using a Beckman Type 70Ti rotor. Solubilization of toxin complex from Super Dome cells was carried out by resuspending the pellet in DPBS (pH 7.0–7.2) containing 137 mM NaCl and 2% (w/v) n-dodecyl-β-d-maltoside (DDM) and incubation at 4 °C overnight, a modification of the method described by Shimada et al.^{34 (link)} where the toxin complex from MDCK cells was solubilized by resuspending the pellet in 20 mM sodium phosphate buffer (pH 7.4) containing 0.05% (w/v) DDM and incubation at 25 °C for 1 h. After incubation, insoluble material was removed by ultracentrifugation at 75,000 × g for 45 min at 10 °C using a Beckman Type 70Ti rotor. Solubilized Etx oligomers were bound to Ni-NTA Agarose resin (Qiagen), washed with 2× 20 volumes of DPBS, 0.02% (w/v) DDM and eluted in three volumes of DPBS, 200 mM imidazole and 0.02% (w/v) DDM.

A combination of ultracentrifugation, iodixanol density cushion, and SEC was used for isolation of larger numbers of EVs of higher purity. Briefly, 40–80 mL of plasma pooled from several individuals was diluted in PBS and centrifuged at 16,500×g_avg (Type 70 Ti rotor, k-factor 950.6, Beckman Coulter) for 20 min to pellet larger EVs such as microvesicles. The supernatant was subjected to ultracentrifugation at 118,000×g_avg (Type 70 Ti rotor, k-factor 133.7, Beckman Coulter) for 2.5 h to pellet smaller EVs such as exosomes. Both EV pellets were re-suspended in PBS and mixed into one sample with a final volume of 6 mL that was loaded onto an iodixanol cushion (as described above). Again, the fraction between the 10 and 30% layer was collected and loaded onto an SEC column, and fractions were collected as described above.