Exoquick exoq5tm 1

Exosomes were isolated and purified using ExoQuick (EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA), Invitrogen Total Exosome Isolation Kit (4484451, Thermo Fisher Scientific, Massachusetts, Waltham, USA), and Exosome-Human CD81 Flow Detection Reagent (10622D, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, the reagents were added to CCM or plasma of healthy donors and cancer patients to isolate exosomes and the mixture was vortexed and centrifuged at 4 °C as described in the manufacturers’ protocols. The pellet containing exosomes was resuspended in DPBS or ultrapure water. Subsequently, the exosome pellet was diluted in M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA) and BCA reagent A and B (A:B = 50:1) was added and incubated for 30 min at 37 °C. The protein concentration of the pellet was determined using the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. To improve reproducibility, all assays were carried out under same experimental conditions.