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Cell confluence is a critical parameter for several reasons. It has a direct impact on transfection efficiency, and on the reproducibility and robustness of the process. It is important to refer to the transfection reagent protocol, as the optimal confluence may differ when comparing several transfection reagents.
For AAV production in adherent HEK-293 cells, we recommend using PEIpro transfection reagent. When using this reagent a confluency of 60-80% at the time of transfection is optimal because cells are still in their exponential growth phase. To improve robustness of the process, think in terms of VCD at the time of transfection and amount of DNA per million of cells to ensure optimal confluency.