Adenovirus Vector Purification Protocol

The E1-deleted Ad5/attP vector was used as wild-type Ad5 in this study (70 (link)). Ad5/attP was propagated in human embryonic kidney–293 cells at 37°C and harvested 36 hours after infection. Subsequently, cells were pelleted by centrifugation, medium was removed, and cell pellets were flash-frozen in liquid nitrogen for storage. Particles were then purified by equilibrium centrifugation in two consecutive CsCl gradients, desalted on a Bio-Rad 10DG column, and stored in Hepes Buffered Saline (HBS), [20 mM Hepes (pH 7.8) and 150 mM NaCl] supplemented with 10% glycerol for long-term storage at −80°C. The data for Ad5-wt have been reported previously (14 (link), 16 (link)).