Computational protocol: Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast

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[…] Temporal analysis of physiological responses of individual cells to stressor exposure was acquired on a BDFACS Canto II (BD Biosciences, San Jose CA, USA) instrument interfaced with FACS Diva v6.11 software (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and analyzed using FlowJo v10.2 software (TreeStar Inc., Ashland, OR, USA). Instrument acquisition, data analysis, and reporting was carried out as suggested by the International Society for Analytical Cytology (ISAC) . The flow rate was adjusted for a maximum of 2000 events per second and assessed by a time versus scatter plot to eliminate artifacts caused by poor flow. Optimal signal to noise ratio was attained by setting detection threshold voltages in the forward scatter (FSC) and side scatter (SSC) channels just below that of the lowermost yeast cell signals. For multicolor flow cytometry non-treated (vehicle), non-strained and single stained controls were used to correctly adjust hardware and software compensation values. For each sample 10,000 events were collected, and a FSC versus SSC (FSC/SSC) plot of non-treated, unstained S. cerevisiae culture for initial gated population P1, to exclude debris in sample analysis. Non-stained and single stained controls were also used for population gating in sample analysis. Each experiment was performed in biological triplicate on independent days. Time points of 1 h, 2 h, 4 h, and 6 h after addition of stressor were evaluated for cell vitality, mitochondrial membrane potential, cellular oxidative state and ROS production, and features of apoptosis. [...] Elemental composition was determined using Agilent 7500 Series ICP-MS instrument and assessed using mixed mode . Wild type BY4741 was grown to stationary phase and stressed with LC50 dosages of hydrogen peroxide and lidocaine, as described above. Aliquots from each culture equaling approximately 106 cells were collected at 1 h, 2 h, 4 h, and 6 h post stress and pelleted at 2500×g for 5 min at 4 °C and washed with 50 mM Tris–HCl pH 7 buffer containing 100 mM NaCl, 5 mM EDTA, and protease inhibitor cocktail (Thermo Scientific). To ensure the washes did not add any ion contamination an empty Eppendorf tube was used as mock sample. The cell pellets were completely dried using a speed vac centrifuge. The whole cell pellets were then reconstituted in 200 μL ICP-MS grade concentrated nitric acid spiked with 50 ppb Gallium as an internal standard and incubated for 1 h at 85 °C and then at room temperature for 4 h. The samples were then diluted 20-fold with 50 ppb Gallium in 1% nitric acid so the final concentration of nitric acid was 5% (v/v) . ICP-MS was performed in biological triplicates. The SEM of all Gallium intensities, was within 5% of the average: with 9 out of the 12 triplicates being ≤1% difference from the average. All ions detected were normalized to Gallium and then to phosphate to compensate for small differences in cell density. In addition, phosphate normalization has been used in cisplatin sensitivity and DNA concentration was shown to be the most accurate normalization biomolecule in metabolomics studies , . Raw data was exported and analyzed using Microsoft excel and SigmaPlot 12.0, and is reported as fold change compared to vehicle-treated control. [...] Metabolites were isolated using a modified Folch extraction method , . A volume equivalent to 5 ODs was centrifuged at 1,500×g for 1 min at 4 °C, washed once with sterile water, and resuspended in 200 μL of methanol. Approximately, 100 μL of acid washed glass beads were added to the cell suspension and vortexed on high for 1 min and then placed on ice for 30 s; this was repeated a total of three times. The suspension containing cellular debris and glass beads was centrifuged again at 4 °C at 15,000×g for 5 min. The metabolite containing supernatant was transferred to a new Eppendorf tube and 400 μL chloroform and 100 μL 5 M NaCl was added. The resulting suspension was vortexed and centrifuged at 1500×g in a table top centrifuge for 5 min. Two-hundred μL of the aqueous phase was collected, transferred to a new Eppendorf tube, and dried down in a speed vac. The sample pellets were then resuspended in 50 μL of LC-grade water and injected onto a 4.6×100 mm Amide X-Bridge (3.5 um, Waters, Milford MA) running at 0.5 mL/min. The gradient consisted of 20 mM Ammonium Acetate, 20 mM Ammonium Hydroxide pH=9.0 (A), and LC-grade acetonitrile (B). The gradient was as follows: 2 min hold after injection at 95% B, followed by a 20 min linear gradient from 95% to 5% B, followed by a 5 min hold at 5% B, and final re-equilibration for  min at 95% B. The ESI Ion-Source was operated at 500 °C, source gasses 1 and 2 at 70 L/min and electrospray potential at 5000 V and -4200 V in positive and negative modes, respectively. The data was collected in positive and negative ion modes with separate injections of 5 μL and 15 μL, respectively. The mass spectrometer was a triple quadrupole 4000 Q-trap (Sciex, Framingham, MA) operating in MRM analysis mode with a dwell time of 10 ms per transition. Analysis of the chromatograms was performed using MetaboAnalyst 3.0, and Microsoft Excel was used for statistical analysis, and Sigmaplot 12.0 () for plotting . All time point samples were analyzed as biological triplicates. […]

Pipeline specifications

Software tools FlowJo, SigmaPlot, MetaboAnalyst
Applications Miscellaneous, Flow cytometry
Organisms Saccharomyces cerevisiae, Homo sapiens
Diseases Multiple Sclerosis, Neurotoxicity Syndromes
Chemicals Adenosine Diphosphate, Adenosine Triphosphate, Amino Acids, Calcium, Carbon, Cysteine, Glutathione, Lidocaine, NADP, Pentosephosphates, Glutamic Acid, Oxaloacetic Acid