Nucleofection of Immortalized Cell Lines

D407 cells (immortalized human retinal pigment epithelial cells) were cultured in DMEM (Cellgro, Mediatech, Inc., Manassas, VA) with 3% FBS (Atlanta Biologicals, Norcross, GA) and 1% Antibiotic/Antimycotic (Cellgro, Mediatech, Inc., Manassas, VA) at 37°C and 5% CO₂. HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% Antibiotic/Antimycotic. HCT116 cells (human colorectal cancer cells) were cultured in McCoy's 5A medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and 1% Antibiotic/Antimycotic. Each nucleofection reaction was comprised of one million cells and 1μg of each plasmid DNA. Nucleofection was performed using the Amaxa Nucleofector (Lonza). D407, HeLa and HCT116 cells were induced for apoptosis using 1 μM staurosporine (STS, Sigma, St. Louis, MO) prepared in DMSO.
D407 cells were a gift from Dr. Aparna Lakkaraju, originating from Dr. R. Hunt at University of South Carolina. HCT116^{BAX-/-/BAK-/-} and HeLa wild type cells were a gift from Dr. Richard Youle [34 (link)]. All cells used in these studies were wild type for endogenous BAX and BAK proteins, unless otherwise stated.