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Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

Livshits MA, Livshts MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM - Sci Rep (2015)

Bottom Line: 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.Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for "fixed-angle" rotors.Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32, Vavilova str., Moscow, 119991, Russia.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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f2: 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).

Mentions: Figure 2a shows the expected proportions of sedimented vesicles of various sizes after 30 min of centrifugation at 10000 g, calculated according to equations (13, 24, 18, 25) for the rotors, listed above. Table 2 represents the corresponding “cut-off” sizes of vesicles d*(30 min, 10000 g) and the proportions of pelleted 150 nm, 120 nm, 100 nm and 70 nm vesicles. The application of the identical protocol for various rotors should result in drastically different size-dependences of pelleting.


Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

Livshits MA, Livshts MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM - Sci Rep (2015)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663484&req=5

f2: 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).
Mentions: Figure 2a shows the expected proportions of sedimented vesicles of various sizes after 30 min of centrifugation at 10000 g, calculated according to equations (13, 24, 18, 25) for the rotors, listed above. Table 2 represents the corresponding “cut-off” sizes of vesicles d*(30 min, 10000 g) and the proportions of pelleted 150 nm, 120 nm, 100 nm and 70 nm vesicles. The application of the identical protocol for various rotors should result in drastically different size-dependences of pelleting.

Bottom Line: 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.Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for "fixed-angle" rotors.Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32, Vavilova str., Moscow, 119991, Russia.

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.

No MeSH data available.


Related in: MedlinePlus