Nucleofection in vitro transfection

R22 cells were transfected using the Amaxa 4D-Nucleofector Optimization Protocol for primary cells (Lonza, Morristown, NJ). R22 cells were grown in culture plates containing DMEM/High glucose supplemented with 10% FBS in a humidified 37 °C/5% CO₂ incubator. Cells were harvested by trypsinization and 6 × 10⁶ cell were resuspended in 82 ul of 4D-nucleofector solution with 18 ul of supplement solution and 6ug of four different plasmids: empty pcDNA (control), Eed-pcDNA, pKdm6b-pcDNA and pEed-pcDNA/Kdm6b-pcDNA. Eed-pcDNA was described previously by us²⁷ and Kdm6b-pcDNA was obtained from Addgene (Plasmid # 24167). Each mix was transferred to a Nucleocuvette and electroporated using the 4D-nucleofector Core Unit (LONZA) using the program CA-137. After the electroporation procedure, cells were incubated at room temperature for 5 min and then transferred to 10 mm culture dishes and incubated for 48 h in DMEM/High glucose media supplemented with 10% FBS. Transfection efficiency was tested using R22 cells electroporated with pEGFP vector. All transfections where performed at three different times and in triplicate.