Neuronal cell viability was determined by fluorescence-based Live/Dead assay (Invitrogen Corporation, Carlsbad, CA) following the manufacturer’s instructions. Briefly, primary cortical neurons were cultured for 14 days on poly-D-lysine-coated 96-well plates followed by treatment with test compounds for 48 h in culture. After two washes with phosphate-buffered saline (PBS), cells were incubated with 2 μM calcein-AM and 4 μM ethidium homodimer (EthD-1) for 20 min at room temperature. Enzymatic conversion of the cell-permeable calcein AM to the fluorescent calcein determined the live cells. Cell death was identified by increased fluorescence resulting from the entry of EthD-1 across damaged cell membranes, and binding to nucleic acids. Using a fluorescence plate reader (Molecular Devices, Sunnyvale, CA), fluorescent calcein was detected at 490 nm excitation and 515 nm emission, while fluorescent EthD-1 was detected at 528 nm excitation and 617 nm emission. The results are represented as percentage of live cells.
