...or would one need to use the combination of the three tetraspanins (CD9, CD63, and CD81) to detect exosomes of different origin? Thanks
Currently we have not a list of EV-specific ‘‘markers’’ that distinguish subsets of EVs from each other.
It's not sufficient find transmembrane protein, because for example it has been demonstrated that CD81 it's not exclusively present on exosomes but also on microvesicles.
So ISEV suggests to demonstrate the presence/absence of three different kinds of proteins on your preparation. In particular, to demonstrate that you are working on exosomes, you should find at least one transmembrane or lipid-bound extracellular protein (CD9, CD63, CD81), because they are enriched or particular present in exosomes; at least one cytosolic protein or endosome/membrane binding protein (TSG101) because they are enriched or particular present in exosomes; and you have to demonstrate the absence or the low presence of intracellular proteins like AGO, HSP90B1 etc, because they are absent or under-represented in exosomes.
So, the combination of at least one of each category allows you to demonstrate that you are working on exosomes.
I would say a combination of TSG101, CD63 and Alix would be beyond doubt to confirm the vesciles are exosomes.
In terms of minimal requirements for exosome definition and function a position paper has been written by Lotvall et al in Journa of Extracellular Vesicles (2014, 3: 26913) "Minimal experimental requirements for definition of
extracellular vesicles and their functions: a position
statement from the International Society for Extracellular
Vesicles". Our experience is that the expression level of e.g. tetraspanins varies a great deal between different types of exosomes (e.g. Oksvold et al. Clinical Therapeutics 2014)
Currently we have not a list of EV-specific ‘‘markers’’ that distinguish subsets of EVs from each other.
It's not sufficient find transmembrane protein, because for example it has been demonstrated that CD81 it's not exclusively present on exosomes but also on microvesicles.
So ISEV suggests to demonstrate the presence/absence of three different kinds of proteins on your preparation. In particular, to demonstrate that you are working on exosomes, you should find at least one transmembrane or lipid-bound extracellular protein (CD9, CD63, CD81), because they are enriched or particular present in exosomes; at least one cytosolic protein or endosome/membrane binding protein (TSG101) because they are enriched or particular present in exosomes; and you have to demonstrate the absence or the low presence of intracellular proteins like AGO, HSP90B1 etc, because they are absent or under-represented in exosomes.
So, the combination of at least one of each category allows you to demonstrate that you are working on exosomes.
agree Philippo. in addition, please be aware that e.g CD9 is not expressed in all exosomes. our findings examining exosomes from different B-lymphoma cell lines demonstrated that all these different types of exosomes did not contain CD9.
its a challenging but very interesting field. so until THE exosome marker "arrives" we will have to combine several different methods and targets to verify the presence of these small vesicles. :)
Actually I isolated exosomes form FaO rat hepatoma cell lines, confirmed exoxomes presence and diameter by electron microscopy. Then I analysed by WB C63 or CD9 and no band could be detected.
I analysed 3 ug of tot proteins, do you think that is too low protein quantity or I should try with another marker?
Grazie
Elena
Can you please provide the reference showing that CD81 is also expressed in microvesicles?
Hello, Audrey,
I found this paper that states, though CD81 are highly expressed in exosomes, it can also be found in other EV fractions but could not be distinguished from exosomes because of a wide heterogeneity in EV size by itself.
Article Tetraspanins in Extracellular Vesicle Formation and Function
Hello Tao Wang,
Do you use reducing or non reducing buffers for CD 81 AND 63?
CD63 is not considered as an ubiquitous marker for all exosomes.
It is cell type dependent. For example, we found that in primary hepatocytes, there is no expression of CD63 at all and CD81 is most expressed and presented on Hepatocute-derived exosomes among the tetraspanin (CD81/9/63)
I also agree with Roi Isaac. CD63 is not considered as an ubiquitous marker for all exosome. its depend on cells. some cells did not express CD63. For your kind information, there are have exosome ELISA kit for all exosome CD9/CD63.
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