For the isolation of EVs, venous blood samples were collected from all study
participants. Whole blood was centrifuged at 13,000 × g for
20 min to isolate plasma. Next, EV isolation and purification from 1 mL of
plasma from each participant was performed using the exoEasy Maxi Kit (#76064;
QIAGEN Inc., Germantown, MD, USA) strictly following the manufacturer’s
instructions. The last step of EV isolation was elution of the EV from the
column. Typically, 400 μL of eluent was obtained.
The isolated EVs were confirmed to be exosomes based on the detection of
tetraspanins, cluster of differentiation (CD)-9, CD-63, CD-81, multivesicular
body synthesis proteins, tumor susceptibility gene (TSG)-101, heat shock protein
(HSP)-70, and the absence of mitochondrial protein and cytochrome c. The size
distribution of isolated EVs was determined by nanoparticle tracking. For the
Western blot analyses, antibodies against CD9 (ab92726; Abcam plc, Cambridge,
UK), CD63 (ab59479; Abcam plc, Cambridge, UK), anti-CD81 antibody (ab109201;
Abcam), TSG-101 (GTX118736; GeneTex Inc., Irwine, CA, USA), cytochrome c
(ab110325; Abcam plc, Cambridge, UK), and HSP-70 (NBP1-77456, Novus Biologicals
LLC, CO, USA) were used at a 1:1000 dilution. Nanoparticle tracking and
characterization were performed using the NanoSight NS300 Instrument (Malvern
Panalytical Ltd, Malvern, UK), following the manufacturer’s instructions.
participants. Whole blood was centrifuged at 13,000 × g for
20 min to isolate plasma. Next, EV isolation and purification from 1 mL of
plasma from each participant was performed using the exoEasy Maxi Kit (#76064;
QIAGEN Inc., Germantown, MD, USA) strictly following the manufacturer’s
instructions. The last step of EV isolation was elution of the EV from the
column. Typically, 400 μL of eluent was obtained.
The isolated EVs were confirmed to be exosomes based on the detection of
tetraspanins, cluster of differentiation (CD)-9, CD-63, CD-81, multivesicular
body synthesis proteins, tumor susceptibility gene (TSG)-101, heat shock protein
(HSP)-70, and the absence of mitochondrial protein and cytochrome c. The size
distribution of isolated EVs was determined by nanoparticle tracking. For the
Western blot analyses, antibodies against CD9 (ab92726; Abcam plc, Cambridge,
UK), CD63 (ab59479; Abcam plc, Cambridge, UK), anti-CD81 antibody (ab109201;
Abcam), TSG-101 (GTX118736; GeneTex Inc., Irwine, CA, USA), cytochrome c
(ab110325; Abcam plc, Cambridge, UK), and HSP-70 (NBP1-77456, Novus Biologicals
LLC, CO, USA) were used at a 1:1000 dilution. Nanoparticle tracking and
characterization were performed using the NanoSight NS300 Instrument (Malvern
Panalytical Ltd, Malvern, UK), following the manufacturer’s instructions.