EVs were isolated by differential ultracentrifugation of CM, according to a protocol (53 (link)), and analyzed as previously described (37 (link)). In brief, cell culture was centrifuged at 300g for 10′ to remove cells, and the supernatant harvested was subsequently centrifuged at 1,800g for 10′ to remove debris, again at 20,000g for 30′, and then at 160,000g for 90′ in the ultracentrifuge (Optima MAX-XP, Beckman Coulter Inc., Irving, TX, USA). All centrifugation steps were performed at 4°C. The pellets were resuspended in phosphate-buffered saline (PBS) without Ca2+/Mg2+ (Sigma-Aldrich) or subjected to protein or RNA extraction.
Pellet particles resuspended in PBS were analyzed for size and concentration by nanoparticle tracking analysis (NTA) using the NanoSight (NS300, Malvern Instruments, Westborough, MA, USA). Samples were diluted in PBS, 300 μl of samples was loaded into the chamber, and five videos for each sample were recorded. Data analysis was done with the NTA software, and data were presented as mean ± standard deviation (SD) of the five videos.
Pellet particles resuspended in PBS were analyzed for size and concentration by nanoparticle tracking analysis (NTA) using the NanoSight (NS300, Malvern Instruments, Westborough, MA, USA). Samples were diluted in PBS, 300 μl of samples was loaded into the chamber, and five videos for each sample were recorded. Data analysis was done with the NTA software, and data were presented as mean ± standard deviation (SD) of the five videos.
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