I tried to isolate exosome from human bone marrow derived mesenchymal stem cell (ATCC-PCS-500-012™) last week. I cultured them very well with DMEM-LG (FBS) or MEM-alpha( FBS). They reached %80 confluency. 24 hrs before I collect the media for exosome isolation, I changed their media with FBS free ones. I applied standard ultracentrifugation protocol for this supernatants. However, I could not see any exosome (or other protein content) when I used nano-drop or BCA kit. When this is the case, I have checked the amount of protein in the supernatant and I found that it is much lower than it should be. What is the reason for this? What am I doing wrong? What is your suggestion?
you can try to isolate exosomes from other "simple" cell lines (eg HeLa) , following your current protocol- and see what you get. You might need to process larger media volume, eg 10 ml - to recover good number of vesicles, that are easy to detect and analyze
You can use media with exosome depleted FBS (available either commercially or made in the lab) for culturing the cell prior to exosome isolation. You may also assess the cell viability prior to collecting the supernatant for exosome isolation.
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