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KMID : 0390320140240010049
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Chungbuk Medical Journal
2014 Volume.24 No. 1 p.49 ~ p.55
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Real-time Monitoring of Hypoxic Cytotoxicity in Neuroblastoma Cells by Using Electric Cell-Substrate Impedance Sensing
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Kim Hoon
Lee Seok-Woo
Park Jung-Soo
Min Jin-Hong
Han Kyu-Hong
Kang Jun-Ho
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Abstract
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Purpose: Cell death from neuronal ischemia-reperfusion injury is a dynamic process. In the minutes to days after an ischemia insult, cellular morphology has been progressively changed. To date, diverse assays, such as LDH assay, MTT assay, PI staining assay et al, have been used for cell viability and toxicity. However, these assays typically require harvesting cells or supernatants at multiple times post-ischemia. Arbitrary harvest intervals post-ischemia are often used based on previous studies or simple feasibility, which may yield to ignore or miss important results taking place between harvest intervals. As a new technique to observe cells in real-time, time lapse microscopy can be alternatively applied to monitor the neuronal ischemic damages. However, it limits quantitative analysis for cellular damages. Electric cell-substrate impedance sensing (ECIS) has been developed for real time monitoring of cell motion and morphological changes through measurements of the changing impedance properties of attached and spread cells that act as insulating particles on gold electrodes It is a technology which can not only produce quantitative data, is also able to monitor experiments in real-time. This study aims to evaluate the ischemia-reperfusion injury by ECIS.
Mathods: Culture dishes were randomly divided into two groups, one in a normoxic incubator and the other in a hypoxic chamber (1% O2: 94%N2: 5%CO2). For OGD insult, cells were washed twice by 1 x D-PBS and added by glucose-free buffer (116 mmol/L NaCl, 1 mM/L NaH2PO4, 5.4 mM/L KCl, 0.8 mM/L MgSO4, 1.8 mM/L CaCl, 26 mM/L Na2HCO3, pH 7.4) and then incubated in a hypoxic chamber for scheduled durations. After scheduled normoxic incubation, cells were assessed for neuronal injury via ECIS Monitoring and MTT assay.
Results: This hypoxia induced SK-N-H cell death in a time-dependent manner. The results obtained from our ECIS system correlated well with conventional in vitro assays such as MTT assays and microscopic findings.
Conclusions: Overall this study illustrates the convenience of ECIS for direct, continuous assessment of the cytotoxicity of Hypoxic injury in vitro. ECIS has the potential to become a useful, non-invasive analytical method for early evaluation of hypoxia injury.
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KEYWORD
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Electric cell-substrate impedance sensing (ECIS), Cell toxicity, Hypoxia
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