Real-Time Detection of Neuronal Cell Death by Impedance-Based Analysis using the xCELLigence System
The xCELLigence System provides a noninvasive and labelfree way to continuously monitor cellular parameters such as viability, death, adhesion, and proliferation. It records electrical impedance of cells grown on microelectrode arrays integrated into the bottom of each well of an E-Plate 96. Cellsensor impedance is expressed as the Cell Index (CI), changes in the CI correlate to modifications in cell morphology. In the study, xCELLigence System's recording of CI values allowed monitoring of primary cortical neuronal culture conditions, including plating, removal of glial cells, compound administration and cell death profiling. In addition, the system clearly revealed neurotoxic and neuroprotective effects in realtime. The system adds significant information to data gained from conventional endpoint assays, identifying optimal time points for performing such assays, while also reducing the time invested in the experiment itself.
Following an initial rise in impedance HT-22 cells displayed a constant growth rate over time with absolute CI values proportional to the initial seeding density, caused by adherence of the cells to the bottom of the well. After treatment with glutamate, CI values began to decrease rapidly, correlating with dosedependent glutamateinduced cell death, which was in turn confirmed by the results of MTT viability assays, as well as being consistent with the kinetics of cell death reported in mitochondrial fragmentation and AIF nuclear translocation assays. In the next step of the experiment, BI-6C9 (an inhibitor of the proapoptotic BH-3 protein BID) was used to prevent glutamate toxicity in HT-22 cells. BI-6C9mediated neuroprotection was reflected in the xCELLigence System by a continued rise in CI values, demonstrating that the compound preserved cell morphology and cell survival.
As with HT-22 cells, primary rat cortical neurons showed at first an increase in CI values due to initial cellular attachment to the bottom of the well. Subsequent removal of proliferating glial cells with cytosinearabinofuranoside caused an expected slight decrease in CI values. To monitor cell death, the primary neuronal cultures were treated with ionomycin or glutamate. Treatment with ionomycin caused a marked drop in CI correlating with the formation of pyknotic, rounded cell bodies. By comparison, cell death induced by glutamate was significantly delayed, and was reflected by a steady decline in CI values within 48 to 72 hours of treatment. These different cell death effects reflect the delayed activation of cell death signalling by glutamate compared to the more rapid loss of cell membrane integrity and necrotic cell death following ionomycin treatment.
(1) S. Diemert, J. Grohm, S. Tobaben, A. Dolga, C. Culmsee (2010) Focus Application Note 6 Neurotoxicity, Roche Applied Science.
Roche Diagnostics Deutschland GmbH
Headquartered in Basel, Switzerland, Roche is a leader in researchfocused healthcare with combined strengths in pharmaceuticals and diagnostics. Roche is the world's largest biotech company with truly differentiated medicines in oncology, virology, inflammation, metabolism and CNS. Roche is also the world leader in invitro diagnostics, tissuebased cancer diagnostics and a pioneer in diabetes management. Roche's personalised healthcare strategy aims at providing medicines and diagnostic tools that enable tangible improvements in the health, quality of life and survival of patients. In 2009, Roche had over 80'000 employees worldwide and invested almost 10 billion Swiss francs in R&D. The Group posted sales of 49.1 billion Swiss francs. Genentech, United States, is a wholly owned member of the Roche Group. Roche has a majority stake in Chugai Pharmaceutical, Japan. For more information: www.roche.com.