Cellbind surface hyperflask m cell culture vessels

Ninety times 10⁶ low-passage-number 293 cells were seeded into 10-layer CellBIND Surface HYPERFlask M cell culture vessels (Corning; 10030) in cDMEM and allowed to adhere overnight at 37°C in 5% CO₂. The next day, 293 cells were transfected with p89.6 DNA by using 25-kDa linear polyethylenimine (PEI; Polysciences, Inc.; 23966) as a carrier and 150 mM NaCl as a diluent. Cells were incubated with the transfection cocktail in the HYPERFlask for 1 day before the medium was exchanged for UltraCULTURE serum-free medium without l-Gln (Lonza; 12-725F) containing 1% HEPES, 1% GlutaMAX (Gibco; 35050-061), and 0.6 mg/ml glucose. The transfected 293 cells were then incubated for an additional 2 days. Viral supernatants were then collected, filtered (Sigma; Z358274-12EA filter), and treated with DNase I (Invitrogen; 18068-015) for 30 min at 37°C to remove any residual plasmid DNA before being aliquoted and stored at −80°C.