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Additional file 2: Figure S2. of The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells

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posted on 24.10.2016, 05:00 authored by Akihisa Okamoto, Masahiro Tanaka, Chisato Sumi, Kanako Oku, Munenori Kusunoki, Kenichiro Nishi, Yoshiyuki Matsuo, Keizo Takenaga, Koh Shingu, Kiichi Hirota
Mitochondrial membrane potential (ΔΨm). Mitochondrial membrane potential was determined by flow cytometry using a MitoPT™ JC-1 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA), according to the manufacturer’s instructions. For these analyses, SH-SY5Y cells were seeded into 6-well plates (3 × 105 cells/well) and cultivated overnight. The following day, cells were treated with the indicated concentrations of the appropriate drug(s) for varying lengths of time and then pelleted by centrifugation at 1200 rpm for 3 min. Supernatants were discharged, and cells were resuspended in JC-1, incubated at 37 °C for 15 min in the dark, and collected by centrifugation at 1200 rpm for 3 min. Supernatants were again discharged and the remaining cell residues were suspended in 500 μl assay buffer. Samples were subsequently analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with CellQuest Pro™ software for the detection of red JC-1 aggregates (590 nm emission) or green JC-1 monomers (527 nm emission). (PDF 2041 kb)


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