3.4. Metabolite Extraction

Metabolites were extracted from GBM and NHA parental cells, media and EVs. Briefly, cell pellets were washed again with 10 mL PBS to remove any residual media and centrifuged at 300× g for 5 min at 4 °C. Cell pellets were held on ice for 5 min to slow down metabolism before being resuspended in 1 mL cold, chilled at −20 °C acetonitrile/water (1:1 v/v) mixture, which further quenches metabolism and lyses cells. Cell suspensions were then centrifuged at 12,000× g for 10 min at 4 °C. The supernatants were dried using a SpeedVac (Freeze dryer FTS Systems, FD-3-85A-MP) overnight at −80 °C. A similar protocol was followed for the extraction of intra-exosomal metabolites, except pellets were resuspended in a 0.2 mL cold acetonitrile/water mixture.