CENTRIFUGATION IN DIFFERENT ROTORS: THEORY

Differential centrifugation, highly used for small and medium EVs isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors.

K-factor vs "cut-off" size

Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for fixed-angle rotors.

The proper centrifugation conditions allowing predicting the size profile of isolated EVs can be selected using theoretical estimates of the “cut-off” sizes of vesicles.

The term “cut-off” means the minimal size of the EVs will be completely sedimented at given centrifugation conditions. For example, after 30 min of 10 000 g centrifugation at 4° C in F-45-24-15 rotor , the cut-off size is 170 nm that means that all vesicles 170 nm and over will be sedimented.

Moreover, for each centrifugation protocol, the portion of sedimented vesicles of given size can be calculated (for example, for the protocol mentioned above, 75% of 100 nm EVs and 37 % of 70 nm EVs will be also sedimented).

This estimates shows that important loss of small and medium EVs could be due to 10 000 g centrifugation in rotor with short sedimentation path length. To increase EVs yield, the centrifugation protocol has to be carefully adapted to the rotor in use.

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