CEVEC: PEI Transfection Video Protocol

Count cells using a hemocytometer or equivalent method to calibrate your counting technique. Start CHO cell culture at a density of 5×10⁵ cells/mL and incubate under standard conditions until reaching 1-5×10⁶ cells/mL.

Dilute your chosen plasmid DNA in Freestyle medium using a sterile reaction tube. Use linearized plasmid DNA for stable transfection of CHO cells or circular plasmid DNA for transient transfection of CHO-T cells.

Dilute polyethylenimine stock solution in Freestyle medium in a sterile reaction tube. Transfer this solution to the tube containing diluted plasmid DNA, vortex briefly, and incubate for 15 minutes at room temperature.

Spin down cells for 5 minutes at 150g and completely aspirate the supernatant to remove any residual medium that could inhibit transfection. Resuspend cells in fresh Freestyle medium and transfer to a 125 mL shake flask.

Add the polyethylenimine-DNA complex solution to the resuspended cells in the shake flask and incubate under standard conditions for 5 hours.

Add four volumes of PM medium containing stable glutamine to the transfected cells. Incubate under standard conditions at 185 RPM for transient expression lasting 7-10 days without requiring medium changes.

Harvest cells by centrifugation for 10 minutes at 150g when optimal viability and protein stability are achieved. This protocol is scalable to higher volumes by maintaining the ratios used for small-scale transfections.