As cell culture media typically contains serum, which includes serum-derived particles that affect EV purity, it is necessary to use serum-free, chemically defined, or EV-depleted medium for collection.
Once cells in the recommended number of culture flasks (20 flasks containing 35 mL of medium each) reach approximately 90% confluency (corresponding to a total culture volume of ~700 mL), wash the cells twice with phosphate‑buffered saline (PBS). The cells are then cultured under serum‑free conditions for 48 hours to allow EV accumulation.
After collecting the conditioned medium, immediately centrifuge it at 800 × g for 30 minutes at 4 °C to remove cells and cell debris.
We recommend shipping the supernatant on the same day as media collection, with morning delivery before 10:30 am preferred.
