Amicon filter tube

PAV variants were produced through a triple plasmid cotransfection of (a) pXX6–80, a helper plasmid encoding adenoviral proteins; (b) pscGFP, self-complementary GFP transgene cassette; and (c) packaging plasmids encoding PAV capsid derivatives.^{17 (link)} Wild-type (wt) capsid AAV2 vector was produced through triple transfection of pXX6-80, pscGFP, and pXX2. Human Embryonic Kidney 293T cells (HEK 293T) were split 1:2 and plated on ten 15 cm plates (BD Falcon) coated with 0.001% poly-L-lysine (Sigma) 24h before transfection. Forty-8 h post-transfection, the producer cells were harvested, pelleted, and resuspended in 1× Gradient Buffer (GB: 10 mM Tris, pH 7.6, 10 mM MgCl2, 150 mM NaCl). Cells were then lysed with three freeze–thaw cycles followed by benzonase treatment (50 units/ml, Sigma) at 37 °C for 40 min. Cell lysate was centrifuged at 4 °C at 3,000g for 20 min and the supernatant was collected for iodixanol density gradient separation (Optiprep, Beckman Ultra-Clear QuickSeal Tubes). Samples were centrifuged at 18 °C at 48,000 rpm for 1h 45 min. Viruses were extracted from the 40% iodixanol layer and stored at 4 °C in 3 mL cryovials (Biotix). Viruses were purified with an anion exchange column (Pall Corporation) and concentrated with an amicon filter tube (Millipore) as published elsewhere.^{17 (link)}