Exosome Isolation and RNA Extraction

Cells were grown in 10-cm plates for 48 hours beforehand, then cells and culture supernatant were collected. The medium was replaced with either “advanced DMEM” (Thermo Fisher Scientific) or RPMI containing an antibiotic-antimycotic mixture and 2 mM L-glutamine (not containing fetal bovine serum), and incubated for 48 hours. Approximately 6 × 10⁴ cells and 1.5 mL cell culture supernatant (into which extracellular particles such as exosomes were released) were collected.
Exosomes were prepared by further extraction from the cell culture supernatant; cells and cell debris were removed by centrifugation at 2,000 × g for 10 minutes at 4°C and filtration, followed by further centrifugation at 110,000 × g for 70 minutes at 4°C. The pellets were washed and resuspended in 11 mL phosphate-buffered saline, and centrifuged again at 110,000 × g for 70 minutes at 4°C [13 (link)]. Finally, the pellet (exosomes) was resuspended in 300 μL phosphate-buffered saline.
Total RNA derived from the cell culture supernatant or exosomes was extracted using the 3D-Gene RNA extraction reagent (Toray Industries, Inc., Japan), whereas total RNA derived from cells was extracted using the miRNeasy Mini kit (QIAGEN, Hilden, Germany, catalog #217004). (dx.doi.org/10.17504/protocols.io.vu3e6yn)