Sw41 ultracentrifuge rotor

Polysome analysis was carried out essentially as described [65 (link)]. Yeast strains were grown in SD medium until mid-log phase was reached, and cycloheximide added to a final concentration of 200 μg mL⁻¹, 15 min. before harvest. Cells were broken in polysome lysis buffer (25 mM Tris pH 7.4, 50 mM KCl, 5 mM MgCl_2, 5 mM 2-mercaptoethanol containing 200 μg mL⁻¹ cycloheximide using glass bead lysis, and a Fastprep bead disruptor machine (MP Biologicals). Following supernatant clarifying centrifuge steps (10 min, 3500× g, 15 min. 15,600× g), lysate was loaded onto a 12 mL 15–50% sucrose gradient made up in polysome lysis buffer, and the gradient spun at 36,000 rpm for 2.25 h using a Beckman SW41 ultracentrifuge rotor.

Cells were seeded in T175 flasks and grown until 70% confluence. Cells were washed in PBS, and all media were replaced with 10 mL of exosome-free DMEM and incubated for 24 h. The medium was removed and centrifuged at 500× g for 10 min to remove cell debris. The supernatant was then filtered through a 0.22 µm filter and ultracentrifuged at 120,000× g for 90 min at 4 °C in an SW41 ultracentrifuge rotor (Beckman Coulter, IN, USA) with swing buckets. The supernatant was removed, and the exosome pellet was resuspended in 1 mL filtered PBS and stored at −70 °C until further use.