You always need to use EV depleted FBS for cell culture if your end point is isolation of EV from those cells.

Hi Mairead,

It depends on the type of flasks that you will be using in order to culture your cells.If you are using plates or generic flasks I would recommend that you use EV depleted FBS for the entire duration of your experiments as you will be certain that your final sample is not contaminated with bovine EVs. However, you are also able to establish your cells in FBS medium, leave them until they reach an appropriate confluency (60-70%), wash 3 times with PBS and replace with the depleted FBS medium until you reach confluency (80-90%).

An article which shows the use of this method has been attached

Article Efficient ultrafiltration-based protocol to deplete extracel...

Another option is that you could use bioreactor tanks in order to culture your cells. This method allows you to culture large amounts of material in a short period of time and use both "normal" FBS and serum-free medium e.g. Insulin Tranferrin Selenium (ITS) simultaneously.

An article which explains the method to use for the bioreactor has been attached.

Article Deep Sequencing of RNA from Three Different Extracellular Ve...

Hope this helps

Thanks @MaryNoel, the feedback is very helpful. I'm growing the cells on plastic and will try washing the cells with PBSx3 before adding EV depleted FBS, if this doesn't work then i'll use the EV depleted FBS from the start.

The articles are very helpful too.

Hi Mairead,

I agree with Mary and I'd also use complete medium to begin and let them grow, then wash with PBS and change to serum-free/EV depleted. Good luck!