Isolation of Microalgae-Derived Extracellular Vesicles

The 50 ml batch‐cultures were centrifuged on day 30 at low speed to separate cells from the culture medium. Then, the separation of microalgae‐derived EVs was performed by differential centrifugation (dUC) (Romancino et al., 2013 (link)). Large EVs (lEVs) were isolated in 50 ml Eppendorf polypropylene conical tubes at 10,000 × g for 30 min at 4°C using an Eppendorf rotor F34‐6‐38. Small EV (sEVs) were then collected from the supernatant into Beckman Coulter polypropylene open top tubes via centrifugation at 118,000 × g for 70 min at 4°C using a Beckman SW28 rotor. After a Phosphate‐buffered saline (PBS 1X, without Calcium and magnesium, Thermo Fisher Scientific) washing step, the pellet was re‐suspended in PBS for subsequent analyses, these sEV preparations are the dUC‐isolated nanoalgosomes.