For getting the results we present in our datasheet, we recommend the follwing strategy: Cells should be stimulated and cultivated for 3 days before Nucleofection, preferentially on plates coated with 1 µg/ml anti-CD3 and 2 µg/ml anti-CD28 antibody. Alternatively, cultivate with 2 µg/ml PHA for 24 hours, wash cells once with medium the following day and add fresh medium supplemented with 25 U/ml IL-2 . Replace medium every 2-3 days and cultivate the cells for 3 days before Nucleofection™ to get high transfection efficiencies.