Hi everyone,
I have been isolating extracellular vesicles from an osteosarcoma cell line for awhile now. I use the ultracentrifugation method (300g 10' + 16500g 20' + syringe filtering of the supernatant + 2x 120000g 70' at 4ºC). After the last ultracentrifugation step, I resuspend the EV pellet in 25ul PBS (I normally begin with 17-18ml of conditioned medium from a T175 cell culture flask).
For EV (total protein) quantification, I use the BCA method. I use 2.5ul of my EV suspension + 2.5ul of PBS + 5ul of SDS 2% (for EV membrane lysis). Due to the very small amount of protein, I use an adapted standard curve obtained from 0, 5, 25, 50, 125 and 250ug/ml of BSA. Usually, the values I get range from 400-600ug/ml (10-15ug of protein/T175 flask ~85/90% confluent).
The thing is the last three times that I tried to isolate EVs the yield that I can get is basically zero! I have prepared fresh EV-depleted medium, the cells look nice and are growing normally, and the BCA method is working properly as there are other people using it in our lab and haven't reported any abnormalities...
Did this happened to any of you before? Or does anyone have a clue of what is might causing these results?
Thank you so much in advance for your help!
Diana
Hi! Are you using any nanoparticle tracking analysis (NTA) such as ZetaView or NanoSight to measure the EVs you are isolating? Or are you using only BCA to measure your EVs by measuring their protein concentrations? We usually should do NTA for EVs concentrations & sizes. If you used to get a good amount of protein from the same cells, your later trials do sound strange. But it could be the number of EVs or the content (cargo proteins) of EVs changing over passages. Or, your EVs could be degrading during the isolation process. Or, you could grow the cells in EV-free media for a little longer & then collect the media for EV isolation.