Effectene transfection protocol

Neuroblastoma 2a (Neuro2a) cells (Olmsted et al., 1970 (link)) were cultivated in DMEM with 2 mM glutamine, 1% non-essential amino acids (NEAA), 1% penicillin and streptomycin (PS), and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded in 24-well plates or glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) and transfected with 200 ng (single transfection) or 400 ng (double transfection) endotoxin-free plasmid DNA, using the Effectene transfection protocol (Qiagen Inc., Valencia, CA, USA). For western blot, whole cell protein lysates were prepared 48 h after transfection. The 20 μg protein was separated by 10% SDS-PAGE, transferred to 0.2 μm Midi format nitrocellulose membrane and processed using the iBind^TM Western System (Bio-Rad Inc., Mississauga, ON, Canada). Primary antibodies were diluted 1:1,000 [mouse anti-GFP, Roche; rabbit anti-GFP (FL), Santa Cruz Biotechnologies, Dallas, TX, USA] and 1:20,000 (mouse anti-β-actin; Sigma-Aldrich Chemie GmbH, Munich, Germany). The secondary antibodies (LI-COR Biosciences, St. Lincoln, NE, USA) were diluted 1:20,000 (donkey anti-rabbit IRDye680LT) or 1:20,000 (goat anti-mouse IRDye800CW). Signals were detected using the Odyssey^® CLx Infrared Imaging System (LI-COR Biosciences).