pcDNA™3.1D/V5-His TOPO®-hsEH

Protocol tips

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	A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific). The cells were cultured for two days at 37 °C, 8% CO2, and 100 rpm, in a shacking incubator, up to a density of 1–3 × 106 cells/mL and 90% viability.

Protocol tips

A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific). The cells were cultured for two days at 37 °C, 8% CO2, and 100 rpm, in a shacking incubator, up to a density of 1–3 × 106 cells/mL and 90% viability.

Publication protocol

A vial containing 1 × 107 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific). The cells were cultured for two days at 37 °C, 8% CO2, and 100 rpm, in a shacking incubator, up to a density of 1–3 × 106 cells/mL and 90% viability. Cells were sub-cultured four times, every two days of incubation, in 30 mL of fresh SFM to 0.2–0.5 × 106 cells/mL. During the fifth and last passage, the cells were seeded in 125 mL of fresh medium to 1 × 106 cells/mL. After 48 h, the cells were prepared for transfection, being resuspended at a cell density of 2 × 106 cells/mL in 250 mL of fresh pre-warmed SFM. The vector and the transfection agent polyethylenimine (Sigma) were added to the cells to a final concentration of respectively 3 μg/mL and 9 μg/mL. After 24 h, the culture was diluted 1:1 in pre-warmed SFM supplemented with 2.2 mM Valproic Acid (Sigma). Three days later the cells were collected by centrifugation at 1200 rpm for 20 min at room temperature and stored at −20 °C.

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