After blood was drawn, serum was immediately separated from the cellular fraction by centrifugation at 3000g for 10 min at ambient temperature and stored at −80°C until use.
Exosomes were extracted from 250-μl serum samples using the ExoQuick kit (SBI, Mountain View, CA, USA) according to the manufacturer's instructions. An aliquot of the exosomes was fixed in Formaldehyde/Glutaraldehyde, 2.5% each in 0.1M Sodium Cacodylate Buffer, pH 7.4 for 15 minutes for electronic microscopy imaging. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp., Danvers, MA) by the High Resolution Electron Microscopy Facility at MD Anderson. The remaining exosome pellets were suspended in PBS and serially diluted to the optimum dynamic range of the Zetaview nanoparticle analyzer (Particle Metrix, Diessen, Germany) for measurement of size and particle density. A separate exosome preparation from 250-μl serum samples was used to examine exosome markers by Western blotting with the following primary antibodies 1:1000 CD63, 1:1000 CD81, and 1:1000 CD9 (CD63, CD81, and CD9 rabbit anti-human from EXO-AB System bioscience). The detailed experimental protocols of these assays have previously been described [31 (link)].
Exosomes were extracted from 250-μl serum samples using the ExoQuick kit (SBI, Mountain View, CA, USA) according to the manufacturer's instructions. An aliquot of the exosomes was fixed in Formaldehyde/Glutaraldehyde, 2.5% each in 0.1M Sodium Cacodylate Buffer, pH 7.4 for 15 minutes for electronic microscopy imaging. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp., Danvers, MA) by the High Resolution Electron Microscopy Facility at MD Anderson. The remaining exosome pellets were suspended in PBS and serially diluted to the optimum dynamic range of the Zetaview nanoparticle analyzer (Particle Metrix, Diessen, Germany) for measurement of size and particle density. A separate exosome preparation from 250-μl serum samples was used to examine exosome markers by Western blotting with the following primary antibodies 1:1000 CD63, 1:1000 CD81, and 1:1000 CD9 (CD63, CD81, and CD9 rabbit anti-human from EXO-AB System bioscience). The detailed experimental protocols of these assays have previously been described [31 (link)].