Lemon nanovesicle (LNV) isolation

Nanovesicles were isolated from Citrus limon L. juice as previously described.⁶ Fruits were obtained from a private farmer, carefully washed in water, and manually squeezed. The juice was sequentially centrifuged at 3,000 × g for 15 minutes, and 10,000 × g for 1 hour. The supernatant was filtered at 0.8 μm and 0.45 μm pore filter and centrifuged at 16,500 × g for 3 hours. Then the supernatant was ultra-centrifuged at 100,000 × g for 105 minutes in a Type 70 Ti, fixed angle rotor, the pellet was washed and suspended in phosphate-buffered saline (PBS). LNVs quantification was determined with the Bradford assay (Pierce, Rockford, IL, USA). On average, from 240 ml of Citrus juice, we recovered 600 micrograms of vesicles suspended in PBS, which correspond to 1.87x10¹¹ particles (3.13x10¹¹ particles/ml).