I performed exosome isolation from cell culture media by using a kit for cell culture media.
Now, I would like to perform the isolation of exosomes inside the cells. Which protocol can I follow?
Maybe you should try an Ultracentrifugation using the cell pellet, after the final 100000xg centrifugation, your pellet should be the Extracellular Vesicles, along with other contaminants like lipoproteins.
Exosomes can be isolated from a cell pellet using a variety of techniques. Ultracentrifugation is one of the most often used techniques, and it entails a series of high-speed centrifugation processes to separate the exosomes from other cellular components according to their size and density.
Here is a general procedure for utilising ultracentrifugation to separate exosomes from a cell pellet:
1 Collect cells and discard culture medium
2 Wash cells in PBS to get rid of any remaining media.
3 Add an adequate volume of PBS or a buffer suited for exosome isolation to the cell pellet
4 Sonication, repeated freeze-thaw cycles, or another suitable technique can all be used to lyse the cells.
5 To remove big cellular debris, centrifuge the lysate at 2,000 x g for 10 minutes.
6 To remove smaller cellular debris and organelles, transfer the supernatant to a fresh tube and centrifuge at 10,000 x g for 30 minutes.
7 Transfer the supernatant to a separate tube and pellet the exosomes by ultracentrifuging it at 100,000 x g for two hours.
8Throw away the supernatant and resuspend the exosome pellet in the proper buffer for further research or storage.
It is necessary to remember that there are other techniques besides ultracentrifugation for isolating exosomes, including precipitation, size-exclusion chromatography, and immunoaffinity capture. The particular study issue, the equipment that is accessible, and the anticipated downstream application all influence the method selection.
that is the best method you can use of ultracentrifugation whit acceleration 100000 gm
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