Figure 1. A typical ultracentrifugation protocol.

In consecutive rounds of centrifugation and pouring off, the RCF (g) and the centrifugation time are increased to pellet smaller particles. After each of the first three centrifugations, pellets that contain dead cells and cell debris are discarded, and the supernatant is kept for the next step. In contrast, after the 100 000 g centrifugations, pellets (containing EVs) are kept, and supernatants are discarded. The pellets are resuspended in phosphate buffered saline (PBS) for further analysis.