title: Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.
abstract: Exosomes, small (40-100 nm) extracellular membranous vesicles, 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. For both types of rotors--"swinging bucket" and "fixed-angle"--we 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, we developed a web-calculator.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Livshits
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32, Vavilova str., Moscow, 119991, Russia.|Moscow Institute of Physics and Technology, 9 Institutskiy Per., Dolgoprudny, 141700, Russia.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Livshts
authors.affiliation: Fededal Research and Clinical Center of Physical-Chemical Medicine, 1a, Malaya Pirogovskaya str, Moscow, 119435, Russia.
authors.forename: Elena
authors.initials: E
authors.lastname: Khomyakova
authors.affiliation: Faculty of Chemistry, Lomonosov Moscow State University, 1/73, Leninskie gory, Moscow, 119991, Russia.
authors.forename: Evgeniy G
authors.initials: EG
authors.lastname: Evtushenko
authors.affiliation: Fededal Research and Clinical Center of Physical-Chemical Medicine, 1a, Malaya Pirogovskaya str, Moscow, 119435, Russia.|Moscow Institute of Physics and Technology, 9 Institutskiy Per., Dolgoprudny, 141700, Russia.
authors.forename: Vassili N
authors.initials: VN
authors.lastname: Lazarev
authors.affiliation: Fededal Research and Clinical Center of Physical-Chemical Medicine, 1a, Malaya Pirogovskaya str, Moscow, 119435, Russia.|Moscow Institute of Physics and Technology, 9 Institutskiy Per., Dolgoprudny, 141700, Russia.
authors.forename: Nikolay A
authors.initials: NA
authors.lastname: Kulemin
authors.affiliation: Research Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, 24 Kashirskoye Shosse, Moscow, 115478, Russia.
authors.forename: Svetlana E
authors.initials: SE
authors.lastname: Semina
authors.affiliation: Fededal Research and Clinical Center of Physical-Chemical Medicine, 1a, Malaya Pirogovskaya str, Moscow, 119435, Russia.
authors.forename: Edward V
authors.initials: EV
authors.lastname: Generozov
authors.affiliation: Fededal Research and Clinical Center of Physical-Chemical Medicine, 1a, Malaya Pirogovskaya str, Moscow, 119435, Russia.
authors.forename: Vadim M
authors.initials: VM
authors.lastname: Govorun
mesh_terms: D000465:Algorithms; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation / Q000379:methods*; D055354:Exosomes; D019073:HT29 Cells; D006801:Humans; D008962:Models, Theoretical