Exosome Isolation and Lipid Extraction
Exosomes were isolated from 100 μL of plasma using Invitrogen total exosome isolation kit (Thermofisher Scientific) according to the manufacturer’s protocol. The isolated exosome pellet was resuspended in 100 μL of ice-cold PBS and dispersed completely by repeatedly pipetting up and down. Following this, 750 μL of ice-cold chloroform: methanol (1:2) (v/v) was added to inactivate the samples. Lipid were then extracted in identical steps as described above for plasma samples, and organic extracts pooled from two rounds of extractions were used for lipidomics analysis. The remaining aqueous/methanol phase containing the extracted pellet was dried in the Speedvac under H2O mode. Proteins were extracted from the dried pellet using RIPA lysis buffer with protease inhibitor cocktail (Sigma-Aldrich), and total protein content was determined using Pierce BCA protein assay kit (Thermofisher Scientific) according to the manufacturer’s instructions.