Start 4D-Nucleofector system and create or upload the file with following experimental parameters:
SE Cell Line solution and 4D-Nucleofector DN-100 program.
 Add 3.6 µl of nucleofector supplement to 16.4 µl of nucleofector solution per sample nucleofected. Alternatively, add entire supplement supplied in the kit to the nucleofector solution, and use 20 µl of this mix per sample nucleofected. When stored at 4°C, the nucleofector solution and supplement mix is stable for 3 months. Thaw plasmid DNA on ice and prepare plasmid mix by adding equal amounts of pCEh-OCT3/4, pCE-hSK, pCE-hUL, and pCE-mp53DD plasmid DNA (250 ng each/sample) in a 0.5 ml tube. Keep the tube on ice. To prepare LCLs for nucleofection, gently pipette LCL culture 3–5 times to make single cell suspension, then collect 5 ml of cell suspension in a 15 ml tube and centrifuge at 500 × g for 5 min at room temperature. Aspirate the supernatant and resuspend cells in 3–5 ml of pre-warmed PBS without CaCl2 and MgCl2. Determine the cell density using preferred cell-counting method and aliquot 4 × 105 cells in a 1.5 ml microcentrifuge tube. Centrifuge the LCL aliquot at 500 × g for 5 min at room temperature. Remove supernatant completely. Add 1 µg of the plasmid DNA mix prepared in step 3.5 and resuspend the cell pellet gently in 20 µl of room temperature nucleofector solution mix prepared in step 3.4. The volume of plasmid DNA mix should not exceed 10% of the total nucleofection reaction volume. Transfer 20 µl of the cells and plasmid DNA mix into a well of 16-well nucleocuvette strip (supplied with nucleofection kit). Gently tap the nucleocuvette strip to make sure the sample covers the bottom of the cuvette. Place nucleocuvette strip into the retainer of the 4D-Nucleofector X Unit and start nucleofection. After run completion, remove the nucleocuvette strip from the retainer, add 80 µl of prewarmed (37°C) RPMI complete medium, and incubate for 5 min at room temperature. Mix cells by gently pipetting 2–3 times and transfer to the 35 mm cell culture dish prepared in step 3.1. Transfer the culture dish to a CO2 incubator at 37°C, 5% CO2 and atmospheric O2 and allow the cells to recover overnight (16–18 h).