Specifically how can I isolate exosome from condition medium and ascites, how to break down exosome and study the composition of membrane (such as lipid, protein) as well as contents (miRNA, DNA). Thank you very much.
Hi! I am no expert in that field, but maybe you can find your answers there:
http://exocarta.org/index.html
You need to culture large number of cells for collection of exosome fractions from media. We do it by growing cells in 100 cm dishes (20 such dishes, each with 10 ml media were used) by seeding 1.5 million cells initially and allowing them to attain 60 percent confluency. Thereafter media is changed with fresh one and cells allowed to grow for 48 hours. After 48 hours, conditioned medium is collected and centrifuged at 500 rpm to pellet down the cell debri. Finally the supernatant from previous step is subjected to ultra centrifugation at 1,20,000 rpm for an hour to pellet down the exosomes. After this, add PBS to the pellet and do an ultra centrifuge again at 1,20,000 rpm., then remove the supernatant. This step removes the contaminating proteins from exosome preparations. The purity of exosomes may be checked by subjecting the pellet for protein extraction and doing a western blot with exosome markers (we do it with CD 63). For protein extraction, you can add any of the lysis buffers like RIPA or NP-40 and for RNA and DNA use trizol.
Hi Asif, Thank you. One question. how would the "After this, add PBS to the pellet and do an ultra centrifuge again at 1,20,000 rpm., then remove the supernatant" step remove the contaminating proteins if super centrifugation was used.
Sorry for the late reply. Actually this is a step to remove contaminating proteins from cell culture media. Adding PBS will help to solubilize them and during addition the exosome pellet may get disrupted. Therefore you need to do one more ultra and finally remove the supernatant (PBS) to get rid of contaminating proteins. Hope this satisfies your query.
I see. makes sense. thank you.
Do you guys know what exosome purification efficiency is generally speaking? I guest it may be low.
Hi Yilin,
The "classic" paper on exosome isolation and characterization is the one from Théry et al. (Isolation and characterization of exosomes from cell culture supernatants and biological fluids), of which I've added the link below to request a full text.
I have recently published another study on isolation methods and the resulting purity myself, where you can also find some protocols for protein and RNA analysis. This paper is open access (second link). Based on these results I would advise to stay away from commercial kits if you can. For the highest purity, go for an OptiPrep or sucrose density gradient.
http://www.journalofextracellularvesicles.net/index.php/jev/article/view/24858#article
Article Current Protocols in Cell Biology
From a more general point of view, here attached are two very interesting articles.
Best regards
Robert
- Badges
- Science method