To isolate EVs from cultured cell lines, the cells were initially rinsed with phosphate-buffered saline (PBS) (Servicebio, #G4202, China) prior to incubation in culture media devoid of EVs. After 3–4 days, the cultured supernatant was collected for further extracting EVs. To separate plasma, whole blood collected in EDTA was centrifugated at 1500g for 10 min at 4 ℃. Tissue explant culture followed a previously described method [41 (link)].
The conditioned medium of either cultured cells, tissue explants, or plasma underwent a series of centrifugations: first at 500g for 10 min, then 3000g for 20 min, and finally 12,000g for 20 min, all at 4 ℃. EVs from the above sources were then obtained by ultracentrifugation at 100,000g for 70 min at 4 ℃, followed by a wash in PBS (Servicebio, #G4202, China) and re-ultracentrifugation. The concentration of EVs was determined using BCA protein assay (Thermo Fisher Scientific, #23225, USA).
The conditioned medium of either cultured cells, tissue explants, or plasma underwent a series of centrifugations: first at 500g for 10 min, then 3000g for 20 min, and finally 12,000g for 20 min, all at 4 ℃. EVs from the above sources were then obtained by ultracentrifugation at 100,000g for 70 min at 4 ℃, followed by a wash in PBS (Servicebio, #G4202, China) and re-ultracentrifugation. The concentration of EVs was determined using BCA protein assay (Thermo Fisher Scientific, #23225, USA).
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