Isolation of exosomes from cell-free plasma

Exosomes were isolated from cell-free plasma using two different methods. In one method Invitrogen Total Exosome isolation (from plasma) kit was used to isolate exosomes following manufacturer’s instructions. Briefly, 0.5 mL of plasma was placed in an Eppendorf tube and mixed with 0.25 mL of PBS. Diluted plasma was then treated with Proteinase K (25 μL) and incubated at 37°C for 10 minutes. Next, each plasma sample was combined with 150 μL of Invitrogen Total exosome isolation (from plasma) reagent and then mixed well by vortexing until a homogenous solution was formed. The samples were incubated at 4°C for 30 min and then centrifuged at room temperature at 10,000 × g for 5 minutes. The supernatant was aspirated and transferred to a new clean tube and stored at -20°C until use. The exosome pellet was re-suspended in PBS buffer and then stored at 4°C short term (1–7 days) or −20°C for long term. The second method used to isolate exosomes was a density gradient centrifugation as previously described by Kalra et al. [7]. First, exosomes were isolated from diluted plasma using a combination of differential centrifugation and ultra-centrifugation as desceribled. The exosome pellet obtained was further purified using density gradient centrifugation. Briefly, a discontinuous OptiPrep™ (60% w/v aqueous iodixanol from Sigma Life Sciences®) gradient consisting of 40% w/v, 20% w/v, 10% w/v, and 5% w/v solutions were prepared in 0.25 M sucrose/10 mM Tris, pH 7.5. The gradient was prepared by layering of 3 mL portions from 40%, 20%, 10% OptiPrep™ solution and finally 2.8 mL of 5% OptiPrep™ solution in a polyallomer tube. Exosome pellet obtained from ultra-centrifugation was layered on top of 5% OptiPrep™ solution and centrifuged at 100,000 × g for 18 h at 4°C. After centrifugation 1 mL gradient fractions were collected from top to bottom and were diluted with 1.5 mL PBS and re-centrifuged at 100,000 × g for 1 h at 4°C. The resulting pellets were characterized by Western blotting and confocal microscopy.