Isolation and Characterization of Extracellular Vesicles from THP-1 Cells

THP-1 cells (10⁶ cells per ml in 150 mm² dishes) were cultured in 15 ml complete medium supplemented with 10% FBS (depleted of bovine EVs by overnight centrifugation at 100,000 g). EVs were isolated from cell culture supernatants via a series of differential ultracentrifugations using a Beckman SW32Ti Swinging Bucket Rotor (Thery et al., 2006 (link)). The final EV pellets were resuspended in PBS. EV protein concentration was measured by standard Bradford dye assay as undertaken previously (Wozniak et al., 2020 (link)). Western blot characterization of the EV preparation has been reported by us previously (Wozniak et al., 2020 (link)) and was performed in this study.