Real-time detection of cellular apoptosis using a rat C6 glioma cell-based assay system
- Authors
- Jung, Kyoung Hwa; Song, Young Me; Das, Nando Dulal; Park, Kyoung Sun; Choi, Mi Ran; Hwang, Sang Youn; Lee, Eun Kyu; Lee, Moon Kwon; Choo, Jaebum; Kim, Kyoung Suk; Kim, Moo Soung; Lee, Sang Rin; Chai, Young Gyu
- Issue Date
- 30-Jun-2011
- Publisher
- SPRINGER
- Keywords
- Caspase-3; Apoptosis; Staurosporine (STP); Time-lapse measurements; Rat glioma C6 cells
- Citation
- MOLECULAR & CELLULAR TOXICOLOGY, v.7, no.2, pp 177 - 184
- Pages
- 8
- Journal Title
- MOLECULAR & CELLULAR TOXICOLOGY
- Volume
- 7
- Number
- 2
- Start Page
- 177
- End Page
- 184
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/46394
- DOI
- 10.1007/s13273-011-0024-y
- ISSN
- 1738-642X
2092-8467
- Abstract
- Caspase-3 is a key mediator of apoptosis in mammalian cells. Cells expressing caspase-3 substrate peptides have become powerful and increasingly common components of cell-based assay systems. We developed a cell-based assay to measure staurosporine (STP)-induced caspase-3 activity in live rat glioma C6 cells. The caspase-3 sensing system was constructed to include a nuclear export signal (NES), followed by the amino acid sequence Asp-Glu-Val-Asp (DEVD) and a green fluorescent protein (GFP) fused to the N-terminal site of a nuclear localization signal (NLS). Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of GFP from the cytosol to the nucleus. After 8 h of 0.5 mu M STP treatment, caspase-3 activity was assessed by monitoring the translocation of GFP to the nucleus due to cleavage of the NES from the GFP by caspase-3. Finally, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. Analysis of caspase-3 activity using real-time monitoring can potentially be used to screen for apoptotic molecules in living cells.
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Collections - College of Natural Sciences > Department of Chemistry > 1. Journal Articles
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