Isolation and Characterization of Extracellular Vesicles

EVs secreted by cultured cells were prepared as previously reported^{10 (link), 11 (link)}. Conditioned media (CM) was first prepared by incubating cells grown at sub-confluence in growth media containing 10% EV-depleted FBS (prepared by overnight ultracentrifugation of medium-diluted FBS at 100,000 ×g at 4ºC) for 48 h, and pre-cleared by centrifugation at 500 ×g for 15 min and then at 10,000 ×g for 20 min. EVs were isolated by ultracentrifugation at 110,000 ×g for 70 min, and washed in PBS using the same ultracentrifugation conditions. When indicated, DiI (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Sigma-Aldrich; St. Louis, MO) was added into the PBS at 1 μM and incubated for 20 min before the washing spin, followed by an additional wash to remove the excess dye. The pelleted EVs were resuspended in ~100 μl of PBS, and subjected to nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern; Westborough, MA), iodixanol/OptiPrep gradient separation, RNA extraction by TRIZOL LS (Thermo Fisher Scientific), and treatment of cells and animals. For gradient separation we used a protocol modified from^{53 (link)–55 (link)}. EVs isolated by ultracentrifugation were loaded onto a 12-step OptiPrep (Sigma-Aldrich) gradient consisted of 30, 27.5, 25, 22.5, 20, 17.5, 15, 12.5, 10, 7.5, 5, and 2.5% iodixanol in 20 mM Hepes (pH 7.2), 150 mM NaCl, 1 mM Na₃VO₄, and 50 mM NaF. After centrifugation in a SW 40 Ti rotor (Beckman Coulter; Indianapolis, IN) at 110,000 ×g at 4°C for 16 h, 12 1-mL fractions were collected and washed in PBS by another spin at 110,000 ×g for 70 min before Western analysis and RNA extraction for RT-qPCR. For cell treatment, 2 μg of EVs (equivalent to those collected from ~5×10⁶ producer cells) based on protein measurement using Pierce™ BCA protein assay kit (Thermo Fisher Scientific) were added to 2×10⁵ recipient cells. Dynasore was obtained from Sigma-Aldrich.