Dear Jenni
Do you want to prepare exo depleted SFB? In that case, using that method, you will get a pellet contained extracellular vesicles but the supernatant will still contain a little amount of exosomes (checked by nanosight). After centrifugation you may use a filter (amicon 100k) for a better depletion.
You need to dilute it in medium (30%FBS). otherwise it is too viscous to centrifuge.
Hi Jenny, as Tom wrote, you have to dilute FBS in medium to centrifuge. I use protocol Thery et al., 2006 (atached file) for preparing exosome production medium and it works well.
prolonged ultracentrifugation is good, typically you should see the exosome/EV pellet. what is dangerous is that at certain point you'll start depleting proteins etc - which are required for cell nutrition. be careful not to compromise cell growth
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