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. 2019 May 14;20(10):2373. doi: 10.3390/ijms20102373

Figure 1.

Figure 1

Schematic representation of the isolation and characterisation of small extracellular vesicles (sEVs) from serum. Serum is centrifuged at 2000× g at 4 °C for 30 min. Pellet is discarded to remove cell contamination (Steps 1 and 2) and supernatant is centrifuged at 12,000× g at 4 °C to remove apoptotic bodies, mitochondrial particles, cell debris and large vesicles (i.e., microvesicles with mean size >200 nm) (Step 3). Supernatant from Step 3 is ultracentrifuged for 2 h at 110,000× g at 4 °C (Step 4) and the pellet is collected (Step 5), resuspended in phosphate-buffered saline (PBS), filtered through a 0.22-μm filter (Step 6) and ultracentrifuged for 70 min at 110,000× g at 4 °C to eliminate contaminant proteins (Step 7). The pellet obtained from Step 7 is resuspended in 100 μL of PBS and represents purified sEVs (Step 8). After isolation, sEVs are analysed to confirm the presence of CD63, CD9 and CD81 markers and their content is characterised via immunoblotting and mitochondrial DNA sequencing analysis (Step 9).