**2.11. Statistical analysis**

loaded with 2 μM Fluo-4AM for 30 min in the presence/absence of ketamine at 37°C followed by a 20-min washout deesterification. The coverslips were placed in a polycarbonate recording chamber (Warner Instruments) on the stage of a laser-scanning confocal microscope. Fluo-4AM fluorescence (λex/λem = 485/520 nm) was visualized using the confocal microscope and the studies were completed in under 8 min to ensure minimal confounding stress to the cells. Fluorescent intensity was then quantified using ImageJ software 1.41 (Wayne Rasband;

hESC-derived neurons cultured on glass coverslips were incubated with 50 nM tetramethylrhodamine ethyl ester (TMRE) (Thermo Fisher Scientific) for 20 min at room temperature. TMRE fluorescent intensity representing ΔΨm was recorded using the confocal microscope (Nikon). Images were obtained from six random fields per coverslip. The data were analyzed

NSCs were cultured in neuronal differentiation medium. Ten days later, the differentiated neurons were transduced with the virus CellLight™ mitochondria-green fluorescence protein (GFP) (Thermo Fisher Scientific) for 24 h to label mitochondria. This fluorescent proteinbased reagent contains the leader sequence of E1-α pyruvate dehydrogenase fused to emerald GFP. The transduced neurons expressed GFP within mitochondria. Four days later, the labeled neurons were used for analysis of the effect of ketamine on the cytochrome c translocation. Virus transduction efficiency was calculated as the ratio between GFP-positive cells and total cells. GFP expression in mitochondria was confirmed by the colocalization of GFP and TMRE fluorescence signals within cells. The distribution of cytochrome c in the neurons was analyzed using immunofluorescence staining with antibody against cytochrome c (Abcam).

hESC-derived neurons were cultured on matrigel-coated plastic coverslips and were exposed to either 20 μM or 100 μM ketamine or control conditions for 24 h. The cells were fixed at 4°C with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer and postfixed for 1 h on ice with 1% osmium tetroxide. The cells were rinsed with distilled water and dehydrated using acetonitrile and graded methanol (50%, 20 min; 70%, 20 min; 95%, 20 min; 100% 3×, 20 min). The cells were embedded in epoxy resin (EMbed-812, Electron Microscopy Sciences, Hatfield, PA) and polymerized at 70°C overnight. Thin (60 nm) sections were cut and the sections were stained with lead citrate and uranyl acetate. The samples were imaged using a Hitachi H600 Electron Microscope.

Following exposure to ketamine or control conditions, total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA). Briefly, the cells were rinsed with PBS and lysed with QIAzol lysis reagent (Qiagen). Chloroform was added and the lysates were centrifuged. The upper phase was

**2.7. Analysis of release of cytochrome c from mitochondria into cytosol**

**2.8. Assessment of mitochondrial shape by electron microscopy**

**2.9. RNA extraction and cDNA preparation**

National Institutes of Health).

426 Mitochondrial Diseases

using ImageJ software.

**2.6. Mitochondrial membrane potential (ΔΨm) assay**

The data presented in this chapter was all collected from at least 3 independent neuronal differentiations. Values were reported as means ± the standard deviation with normal distributions. Statistical analysis was completed using the Student's t-test when comparing 2 groups. All statistical analysis was performed using the SigmaStat 3.5 software (Systat Software, Inc., San Jose, CA).
