**2.4. Assessment of cell death by TUNEL staining**

Apoptosis of the hESC-derived neurons was assessed using a cell death detection kit (Roche Applied Bio Sciences, Indianapolis, IN) based on terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate in situ nick end labeling (TUNEL) following directions provided by the manufacturer. This kit identifies single or double-stranded DNA breaks by labeling the free 3′-OH termini with altered nucleotides in a reaction with terminal deoxynucleotidyl transferase (TdT). Cells cultured on glass coverslips were exposed to ketamine or control conditions, rinsed and fixed with 1% paraformaldehyde. DNA fragmentation was analyzed using TdT, which incorporates into sites of DNA breaks. The nuclei were stained with Hoechst 33342 and the cells were imaged using the confocal microscope. Apoptosis/ necrosis was quantified by assessing the ratio of TUNEL-positive nuclei to total cell nuclei in a field.
