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First and foremost, think in terms of viable cell density rather than absolute surface area when optimizing a transfection process. In other words, calculate the amount of DNA and transfection reagent per million of cells. Be sure to characterize how the cultures grow (e.g., cell yield, doubling time) in the scaled-up vessel where you are conducting the transfection for an accurate assessment of cell density on the day of transfection. If possible, seed a control vessel with the same growth kinetics to harvest on the day of transfection to determine cell density. Normalizing for cell growth in this way can account for inconsistent cell growth between different sizes and/or types of cell culture vessels and ensures reproducible transfections upon scaling-up. It is equally important that the transfection reagents are designed for scalable transfections, like those from Polyplus Transfections, that enable robust transfection complex formation from small scale-process development through intermediate to large scale production.