Vented erlenmeyer flasks

Human embryonal kidney HEK293 F cells were maintained in FreeStyle 293 Expression medium (Gibco, Thermo Fisher Scientific) in vented Erlenmeyer flasks (Corning, Thermo Fisher Scientific) at 120 rpm, in a 37 °C, 5% CO₂, humidified incubator. Cells were transiently transfected in flasks using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s manual. The days before transfection, cells were seeded in fresh culture medium at 0.5 × 10⁶ cells/mL. When the density reached approximately 1 × 10⁶ cells/mL the cells were transfected. DNA was added to Lipofectamine 2000 in a 1:2.5 ratio, incubated at room temperature for 25 min and added to the cells. Supernatants were collected 3 days post transfection by centrifugation at 1500× g, 10 min at 4 °C. cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics) was added to the cleared supernatant prior to storage at −20 °C.

For protein expression, human embryonal kidney HEK293 F cells were maintained in FreeStyle™ 293 Expression medium (Gibco®, Thermo Fisher Scientific) in vented Erlenmeyer flasks (Corning) at 120 rpm, in a 37 °C, 5% CO2, humidified incubator. Cells were transiently transfected in flasks using Lipofectamine 2000 (LifeTechnologies) according to the manufacturer’s manual. 1 or 2 days before transfection, cells were seeded in fresh culture medium at 0.5 × 10⁶ cells/mL. When the density reached approximately 1 × 10⁶ cells/ml the cells were transfected. DNA was added to Lipofectamine 2000 in a 1:2.5 ratio, incubated at room temperature for 25 min and added to the cells. Supernatants were recovered 3 days post transfection by centrifugation at 1500 × g, 10 min at 4 °C. cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics) was added to the cleared supernatant prior to storage at −20 °C.