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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

Comparison of the “general protocol” centrifugation strategy with that using a “cut-off-size” based adjustment of the centrifugation duration for three different rotors.(a) the NTA size distributions changed because of the 30 min centrifugation at 10000 g in the three rotors: (Beckman Type 60 Ti, Eppendorf F-45–24–15 and Beckman SW28); (b) the NTA size distributions after centrifugation with the duration adjusted to the complete sedimentation of 150 nm vesicles and larger; (c) the histograms of the total vesicle concentrations.
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f4: Comparison of the “general protocol” centrifugation strategy with that using a “cut-off-size” based adjustment of the centrifugation duration for three different rotors.(a) the NTA size distributions changed because of the 30 min centrifugation at 10000 g in the three rotors: (Beckman Type 60 Ti, Eppendorf F-45–24–15 and Beckman SW28); (b) the NTA size distributions after centrifugation with the duration adjusted to the complete sedimentation of 150 nm vesicles and larger; (c) the histograms of the total vesicle concentrations.

Mentions: Using three different rotors – two FA-type rotors (Beckman Type 60 Ti and Eppendorf F-45–24–15) and one SW-type rotor (Beckman SW28), we compare the experimental results of the traditional “general protocol” strategy and those obtained with the suggested “cut-off-size”-based adjustment of centrifugation duration. Figure 4a shows the NTA PSD of the initial pre-purified exosomes (red curve) and the PSDs obtained after 30 min of 10000 g centrifugation in SW28, Beckman Type 60 Ti and Eppendorf F-45–24–15 rotors (blue, magenta and green curves correspondingly). The distributions differ substantially. Therefore, different vesicle populations are obtained by equally long centrifugations in different rotors. By contrast, Fig. 4b shows that when the centrifugation durations for different rotors are selected as those corresponding to the complete sedimentation of vesicles of a definite size (“cut-off-size”), the differences in the NTA distributions of vesicle populations in different rotors diminish. Figure 4c displays the histograms of the vesicle concentrations and demonstrates the efficiency of the “cut-off-size”-based “equalization” of the sedimentation abilities of various rotors.


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)

Comparison of the “general protocol” centrifugation strategy with that using a “cut-off-size” based adjustment of the centrifugation duration for three different rotors.(a) the NTA size distributions changed because of the 30 min centrifugation at 10000 g in the three rotors: (Beckman Type 60 Ti, Eppendorf F-45–24–15 and Beckman SW28); (b) the NTA size distributions after centrifugation with the duration adjusted to the complete sedimentation of 150 nm vesicles and larger; (c) the histograms of the total vesicle concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Comparison of the “general protocol” centrifugation strategy with that using a “cut-off-size” based adjustment of the centrifugation duration for three different rotors.(a) the NTA size distributions changed because of the 30 min centrifugation at 10000 g in the three rotors: (Beckman Type 60 Ti, Eppendorf F-45–24–15 and Beckman SW28); (b) the NTA size distributions after centrifugation with the duration adjusted to the complete sedimentation of 150 nm vesicles and larger; (c) the histograms of the total vesicle concentrations.
Mentions: Using three different rotors – two FA-type rotors (Beckman Type 60 Ti and Eppendorf F-45–24–15) and one SW-type rotor (Beckman SW28), we compare the experimental results of the traditional “general protocol” strategy and those obtained with the suggested “cut-off-size”-based adjustment of centrifugation duration. Figure 4a shows the NTA PSD of the initial pre-purified exosomes (red curve) and the PSDs obtained after 30 min of 10000 g centrifugation in SW28, Beckman Type 60 Ti and Eppendorf F-45–24–15 rotors (blue, magenta and green curves correspondingly). The distributions differ substantially. Therefore, different vesicle populations are obtained by equally long centrifugations in different rotors. By contrast, Fig. 4b shows that when the centrifugation durations for different rotors are selected as those corresponding to the complete sedimentation of vesicles of a definite size (“cut-off-size”), the differences in the NTA distributions of vesicle populations in different rotors diminish. Figure 4c displays the histograms of the vesicle concentrations and demonstrates the efficiency of the “cut-off-size”-based “equalization” of the sedimentation abilities of various rotors.

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