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Isolation of extracellular vesicles: Determining the correct approach (Review).

Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M - Int. J. Mol. Med. (2015)

Bottom Line: Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation.The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches.The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Immunology and Transplantology, Jagiellonian University Medical College, 30-663 Krakow, Poland.

ABSTRACT
The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

No MeSH data available.


Related in: MedlinePlus

Differential centrifugation scheme, including a sucrose, iodixanol gradient/cushion step.
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f2-ijmm-36-01-0011: Differential centrifugation scheme, including a sucrose, iodixanol gradient/cushion step.

Mentions: This is probably the most commonly used method for EV isolation/purification (20,30,38). As with other methods, there are variations depending on the laboratory setting; however, for the most part, the protocols follow the scheme that was put forward in the study by Raposo et al, who purified exosomes from the conditioned culture media of transformed human B cell lines (38). The protocol involves a number of sequential centrifugation steps at different centrifugal forces (g) whose purpose is to remove unwanted components from the actual exosomes. The first three steps of the protocol are designed to remove intact cells, dead cells or cell debris using three different centrifugal forces, that is 300 × g for 10 min, 2,000 × g for 10 min and 10,000 × g for 30 min, respectively. After each centrifugation, the supernatant is transferred into a new test tube while the generated pellets are being discarded. After the 10,000 × g spin, the supernatant is then subjected to a final ultracentrifugation at 100,000 × g for 70 min. The outcome of this step is an exosome pellet that can be used for further studies. It should be also noted that all the centrifugation steps are being carried out at 4°C. The basic scheme for EV isolation is presented in Fig. 2.


Isolation of extracellular vesicles: Determining the correct approach (Review).

Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M - Int. J. Mol. Med. (2015)

Differential centrifugation scheme, including a sucrose, iodixanol gradient/cushion step.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494580&req=5

f2-ijmm-36-01-0011: Differential centrifugation scheme, including a sucrose, iodixanol gradient/cushion step.
Mentions: This is probably the most commonly used method for EV isolation/purification (20,30,38). As with other methods, there are variations depending on the laboratory setting; however, for the most part, the protocols follow the scheme that was put forward in the study by Raposo et al, who purified exosomes from the conditioned culture media of transformed human B cell lines (38). The protocol involves a number of sequential centrifugation steps at different centrifugal forces (g) whose purpose is to remove unwanted components from the actual exosomes. The first three steps of the protocol are designed to remove intact cells, dead cells or cell debris using three different centrifugal forces, that is 300 × g for 10 min, 2,000 × g for 10 min and 10,000 × g for 30 min, respectively. After each centrifugation, the supernatant is transferred into a new test tube while the generated pellets are being discarded. After the 10,000 × g spin, the supernatant is then subjected to a final ultracentrifugation at 100,000 × g for 70 min. The outcome of this step is an exosome pellet that can be used for further studies. It should be also noted that all the centrifugation steps are being carried out at 4°C. The basic scheme for EV isolation is presented in Fig. 2.

Bottom Line: Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation.The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches.The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Immunology and Transplantology, Jagiellonian University Medical College, 30-663 Krakow, Poland.

ABSTRACT
The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this reveiw was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this 'uniqueness' is the first step in the right direction for the complete assessment of EVs.

No MeSH data available.


Related in: MedlinePlus