Each transfection reaction was prepared by adding 2 µl of “primed” cells resuspended in SF buffer to a mixture of 14 µlvof SF buffer; 2 µl of 20 µg/µl pUC19; 1 µl of 250 mM ATP, pH 7.5; 1 µl of 100 mg/ml sodium heparin; and =7 µl of reporter DNA. (Note that higher volumes of nucleofection lead to lower transfection frequencies; thus, reporter DNA should be as concentrated as possible, not exceeding 7 µl. Also, see Note about titrating reporter plasmids.) The transfection reaction was transferred to one well of a 96-well nucleofection plate (Lonza; Cat. No. V4SC-2960) or a 16-well strip (Lonza; Cat. No. V4XC-2032). The nucleofection plate was placed in a 96-well shuttle device (Lonza; Cat. No. AAM-1001S) or X-unit (Lonza; Cat. No. AAF-1002F) connected to a Nucleofector 4D core unit (Lonza; Cat. No. AAF-1002B), and the CM156 pulse was applied to each well.
Immediately after pulsation, 100 µl of ice-cold recovery buffer (10 mM HEPES-KOH, pH 7.5; 0.9 M sorbitol; 8% [wt/vol] PEG 8000) was added to the cells. Recovery buffer was gently mixed with the transfected cells by firmly tapping the side of the plate and then incubating the samples for 5 min. The whole volume of the transfection reaction plus the recovery buffer was transferred to 1 ml of 1× High Nutrient Medium in a 12-well plate