Sorvall rc 5b centrifuge

EVs were isolated from 24–48 h conditioned media from breast cancer cell cultures with >95% cell viability, using a sequential centrifugation technique [11 (link)]. Briefly, conditioned media were centrifuged at 400× g for 10 min, 2000× g for 15 min, and 15,000× g for 30 min at 4 °C (Sorvall^® RC-5B centrifuge, Thermo Fisher Scientific Inc.) followed by ultracentrifugation at 100,000× g for 90 min at 4 °C (Optima XE-90 Ultracentrifuge, Beckman Coulter Life Sciences). EV pellets were washed at 100,000× g for another 90 min and were resuspended in PBS.
EV preparations were characterized according to the guidelines of the International Society for Extracellular Vesicles [16 (link)] and as described previously by us [11 (link)]. EV size and concentration was measured by nanoparticle tracking analysis (NanoSight NS300, Malvern Instruments, UK). EV markers were evaluated by western blot and the shape of the EVs was evaluated by electron microscopy [11 (link)].
To isolate TdTomato-labeled EVs, we transduced breast cancer cells with a lentiviral vector to express palmitoylated TdTomato (PalmtdTomato) [53 (link)]. The DNA construct was a gift from Dr. X. Breakefield, Massachusetts General Hospital. The fluorescence label of the isolated EVs were evaluated by fluorescent microscopy and plate reader (SpectraMax M2 plate reader, Molecular Devices LLC, San Joses, CA, USA).

Fresh faeces were collected from the ground immediately after defecation was observed. Faecal samples were stored in screw-top 100 mL plastic containers and stored at −18 °C until processing. Samples were defrosted at room temperature and mixed with a protease inhibitor (cOmplete™, EDTA-free Protease Inhibitor Cocktail, Roche, Germany) at a ratio of 1:1–1:2 (w:v) with phosphate-buffered saline, as per the manufacturers protocol, depending upon consistency and moisture. This mixture was homogenised and centrifuged at 3–6 °C and 12,000×g (Sorvall SLA-3000 rotor in a Sorvall RC-5B centrifuge, ThermoFisher Scientific, Waltham, MA, USA) for 5 min. The faecal supernatant was then removed by pipette and stored at −18 °C.