ufacturer’s directions, and all experiments had been carried out making use of cells from passages five to 20. For experiments, the cells were cultured in 96-well plates precoated with 20 g/ ml laminin, at a density of 2 103 cells per nicely. Cells have been cultured for 2 days in ReNcell NSC Maintenance Medium (Millipore) with 20 ng/mL fibroblast growth aspect (Millipore) and 20 ng/mL epidermal development aspect (Millipore), then cultured for 3 days in ReNcell NSC Upkeep Medium devoid of neuronal differentiation growth aspects.
The expression of neuronal marker class III beta-tubulin and dopaminergic cell marker tyrosine hydroxylase (TH) was assessed in iPSC-derived neurons cultured on glass bottom plates (-Slide eight nicely, Ibidi, Martinsried, Germany) precoated as described above. Right after 14 days of culturing for differentiation, the cells have been fixed with 4% paraformaldehyde for ten min, permeabilized for 5 min with 0.1% Triton X-100, incubated in blocking buffer [1% bovine serum albumin (KPL, Gaithersburg, MD, USA) in PBS] for 1 h, and stained overnight with major antibodies against neuron-specific class III beta-tubulin, (1:500; BioLegend Japan, Tokyo, Japan) or TH (1:250; Abcam, Cambridge, UK), followed by incubation using the secondary antibodies Alexa Fluor 488 Chicken anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 647 anti-rabbit IgG (Invitrogen) for 1 h. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, 1:500; Molecular Probes, Carlsbad, CA, USA) for 1 h at space temperature. Photos have been obtained by a confocal microscope (TCS SP8, Leica microsystems, Tokyo, Japan). The positivity of class III beta-tubulin and TH was measured in 3 separate cultures of each Acetovanillone experimental condition, and five fields in every culture have been analyzed. We also examined the positivity of class III beta-tubulin in the cortical neuronal cell line soon after differentiation as described above. Right after differentiation, the iPSC-derived neurons have been treated with escalating doses (20, 100, 500 M) of ketamine (Daiichi Sankyo, Tokyo, Japan) for six and 24 h, to examine whether ketamine neurotoxicity is time- and/or dose-dependent.
Cell viability evaluation was incorporated in ApoTox-Glo Triplex Assay kit (Promega, Madison, WI, USA). Glycyl phenylalanyl-aminofluorocoumarin, a fluorogenic, cell-permeant, peptide substrate was added to assess cell viability after 6 or 24 h of therapy with ketamine following the manufacturer’s protocol. The substrate enters intact cells, where it can be cleaved by 
the live-cell protease activity to create a fluorescent signal proportional towards the quantity of living cells. Cell viability was assessed by measuring fluorescence using a GloMax Microplate Reader (Promega), applying an excitation wavelength of 400 nm and an emission wavelength of 505 nm.
ApoTox-Glo Triplex Assay (Promega) was utilised for assessing caspase 3/7 activity in neurons. Just after the measurement of cell viability described above, the caspase-Glo 3/7 reagent was added into each well, and the plates had been briefly mixed by an orbital shaker and incubated for 30 min at 37. Within this assay, activated caspases cleave a luminogenic peptide substrate, which releases a luminescent signal by a luciferin/luciferase reaction. Caspase activation was determined by measuring luminescence using a GloMAX Microplate Reader.
ROS-Glo H2O2 Assay (Promega) was applied to measure alterations in the degree of ROS by straight detecting H2O2 in neurons. The cells were plated in white, clear-bottom 96-well tissue