Isolation and Purification of ULF-EVs

ULF-EVs were extracted from ULF samples by employing OptiPrep™ density gradient ultracentrifugation (ODG UC) [15 (link)] as described previously. Briefly, the samples were centrifuged at 10,000×g for 30 min to remove macroparticles and apoptotic bodies and then ultracentrifuged at 100,000×g for 2 h using an SW41T rotor (Beckman Coulter Instruments, Fullerton, CA, USA) to precipitate ULF-EVs twice. The final pellet was resuspended in 100 µL phosphate-buffered saline (PBS) (Gibco, USA). All centrifugation processes were conducted at 4 °C.
Discontinuous iodixanol gradients of 5%, 10%, 20%, and 40% iodixanol were formed in polyallomer tubes by diluting OptiPrep™ (60% (w/v) aqueous iodixanol solutions) with an appropriate amount of 0.25 M sucrose and 10 M tris as previously described (Beckman Coulter Instruments, Fullerton, CA, USA) [16 (link)]. The pellet was then covered and centrifuged for 18 h at 100,000×g at 4 °C (Beckman Coulter Instruments, Fullerton, CA, USA). The 12 stratification fractions were collected and diluted in PBS to eliminate any remaining OptiPrep™, and each fraction was ultra-centrifuged for 3 h at 100,000×g and 4 °C. The pellet was re-suspended and stored at − 80 °C for subsequent tests, and ULF-EVs were primarily in the layer between fractions 7 ~ 11.