I have been trying to isolate exosomes from HEK293 cells. and i have read and tried several protocols. but still can't get any exosomes pellet in the final step. How long should i culture cells in serum-free media? Does longer incubation increases yield of exosomes? if someone can provide me with reliable protocol, that would be really helpful.
Exosome free FBS containing culture media for 72 hours works well. I isolated exosome from HT29 and PANC1 cells, you may try.
Usually, studies use EV depleted FBS but haven't seen serum free. Also, pellet will probably not be visible in any case especially if you don't culture for long, but it is there (re-suspend where it should be).
better use exosome-depleted FBS, eg from Gibco. serum free is tricky
I would go with 48-72h in SFM. Be careful with long exposures with SFM, as cells get affected and stressed, and the exosomes you'll be collected will come from unhappy cells. Either way, keep in mind that the exosome pellet you will obtain is often too small to see.
Best of luck.
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