**2.5. Calcium imaging**

Neurons were plated on matrigel-coated glass coverslips for the calcium imaging studies. Intracellular calcium was assayed using Fluo-4AM (Thermo Fisher Scientific). Neurons were 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; National Institutes of Health).

removed and RNA was precipitated from it with 100% ethanol. The RNA was collected using the RNeasy spin columns (Qiagen). The samples were rinsed with buffer RPE. To elute the RNA from the column, 30 μL of RNase-free water (Qiagen) was added. An Epoch nanodrop spectrophotometer (BioTek Instruments Inc., Winooski, VT) was used to assess the quantity and quality of the RNA in each sample. Each RNA sample was then diluted to 100 ng/μL in RNase-free water. The RNA was reverse transcribed to cDNA using the miScript II RT kit (Qiagen) following the manufacturer's directions. Briefly, a mixture containing 1 μg of RNA, RNase-free water, 10× miScript nucleics mix, reverse transcriptase mix and 5× HiSpec buffer (Qiagen) was prepared. The RT reaction mixture was incubated for 1h at 37°C and for 5min at 95°C to stop the reaction. The RT product was diluted in 200 μL in RNase-free water which yielded a final RNA concentration of 4.5 ng/μL

Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly…

http://dx.doi.org/10.5772/intechopen.72939

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**2.10. Quantitative reverse transcription-PCR (qRT-PCR) analysis of MicroRNAs**

To screen for potential microRNAs contributing to the ketamine-induced neurotoxicity, we used human miFinder miRNA PCR arrays (Qiagen) which screen 84 microRNAs. For the arrays, a master mix (25 μL/well) containing the template cDNA (4.5 ng/well), RNase-free water, universal primer and miScript SYBR Green (Qiagen) was prepared according to the manufacturer's instructions. The primers for each microRNAs to be analyzed come lyophilized in the wells of the array plates. To confirm the array results, validation assays were performed in which the three cDNA samples used for the arrays were each run in triplicate on the same PCR run. The PCR was run using a BioRad iCycler for 15 min at 95°C followed by 40 cycles of: denaturation (15 s at 94°C), annealing (30 s at 55°C) and extension (30 s at 70°C). Melt curve and reverse transcriptase controls were run to ensure sample purity and primer specificity, respectively. The epoch nanodrop spectrophotometer was also used to assess RNA quality using the A260:A280 ratio. To be considered pure, the A260:A280 for an RNA sample must fall between 1.8 and 2.2. Samples with an A260:A280 ratio outside of this accepted range were not used for the studies. MicroRNAs that displayed a 1.3 fold change in expression between the ketamine and control treated cells were considered to be of interest.

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.,

To generate neurons from the hESCs, the cells were taken through a four-step differentiation protocol as outlined in **Figure 1A** and described previously [20, 37, 38]. The neurons

in each sample.

**2.11. Statistical analysis**

**3. Results and discussion**

**3.1. Neuronal differentiation and characterization**

San Jose, CA).
