You’ve been doing your exosome isolation all wrong

Exosomes attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors.

Researchers from the Russian Academy of Sciences express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, the researchers developed a web-calculator.

exosome rna

Expected vesicle size-dependent extents of pelleting by centrifugation at RCF = 10000 g with the rotors specified in the insets. (a) equal centrifugation time lengths (30 min); (b) the time lengths are adjusted according to the “K-factor rule”; (c) time lengths of the centrifugation correspond to a definite size (150 nm) of complete sedimentation (“cut-off-size” rule).

Availability – the interactive web-calculator available at:

Livshts MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM. (2015) Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci Rep 5:17319. [article]

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