Exosome Isolation from HepG2 Cells

HepG2 cells were cultured in 25cm² flasks. When the confluency reached 60-70%, the cells were washed by PBS twice and counted, and the medium was changed to fresh serum-free DMEM with or without TAPI-2 (Sigma) at the final concentration of 25 μmol/L 18 (link). After 24h, the culture supernatants were collected and concentrated by ultrafilters (Millipore) with molecular weight cut-offs of 10,000. The exosomes were prepared from the concentrated supernatants using the ExoQuick-TC^TM Exosome Precipitation Solution (System Biosciences, USA) as described by the manufacturer 7 (link). Meanwhile, the cells were scraped and lysed by cold lysis buffer (Thermo Fisher) with protease inhibitor cocktail (Sigma).