OptiPrep density gradient centrifugation

Five hundred microliter of serum was centrifuged at 12000 xg for 30 min and the supernatant was filtered through 0.22 μm PVDF filter. The filtered sample was diluted with 11 mL of PBS and centrifuged at 110,000 xg for 70 min. The pellet was resuspended in 500 μL of PBS. A stock solution of OptiPrep (60% w/v iodixanol) (Axis-Shield, Dundee, Scotland) was diluted with 0.25 M sucrose, 10 mM Tris-HCl (pH 7.6) to generate 40%, 20%, 10% and 5% w/v iodixanol solutions. A discontinuous density gradient was generated by sequential layering of 3 mL each of 40, 20 and 10% (w/v) iodixanol solutions, followed by 2.5 mL of 5% iodixanol solution in ultracentrifuge tubes. Sample was overlaid on the discontinuous iodixanol gradient followed by centrifugation at 110,000 xg for 16 h. One milliliter fractions were collected from the top of the gradient. Each sample was diluted with 11 mL of PBS and centrifuged at 110,000 xg for 70 min. The pellet was resuspended in PBS and stored at 4 °C until use.