Total exosome isolation from cell culture media kit

For the purification of the exosomes, plates containing cells grown to a confluence of between 50 and 60% were used, with the culture medium being eliminated and replaced by an equivalent volume where the FBS was replaced by an exosome-depleted FBS (Gibco, Waltham, MA, USA). After 72 h of incubation, the culture medium was collected, and the exosomes were obtained. For the characterization of the nanovesicles by nanoparticle tracking analysis (NTA) or electron microscopy, the exosomes were purified using the total exosome isolation from cell culture media kit (Invitrogen, Waltham, MA, USA), following the manufacturer’s instructions. The exosomes destined for peptide analysis by LC-MS/MS were purified using Exo-spinTM mini columns (CELL guidance systems, Cambridge, UK) following the manufacturer’s specifications; in this case, the precipitation step prior to addition to the column was replaced by ultracentrifugation at 110,000× g for 2.5 h to avoid the precipitant interfering with the mass spectrometric analysis. For the isolation of total RNA, the ExoQuick-TC (System Biosciences, Palo Alto, CA, USA) was used, according to the manufacturer’s instructions. Once purified, the exosomes were quantified by determining the protein concentration with the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s instructions.

The exosomes were extracted with the Total Exosome Isolation (from cell culture media) kit (Invitrogen, Carlsbad, CA, USA), according to the retailer protocols, in sterile conditions. After harvesting, the cell culture media was centrifuged at 2000× g for 30 min to remove cells and debris and, then, the supernatant was transferred to a new sterile tube. The required volume of the Total Exosome Isolation reagent was added and mixed with the medium by vortexing until a homogeneous solution was obtained and the tube was incubated in the fridge overnight. Successively, the mixture was centrifuged at 10,000× g for 1 h at 4 °C, the supernatant was discarded and the pellet-contained exosomes were resuspended in 200 μL of sterile water. The exosomes were then ready for their characterization, protein extraction or incubation with cells. The extracted exosomes were stored at −80 °C until proteins analysis was performed.