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Simultaneous analysis of cell death mechanisms and oxidative stress using live cell fluorescence microscopy
Author(s) -
Yan Michelle,
Clarke Scott T,
Huang ShihJung,
Singh Upinder,
Janes Michael S,
Dolman Nicholas J
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.943.21
Subject(s) - programmed cell death , oxidative stress , autophagy , apoptosis , microbiology and biotechnology , cell , biology , caspase , necrosis , oxidative phosphorylation , chemistry , biochemistry , genetics
Cell death can occur through multiple pathways, such as apoptosis, autophagy, and necrosis. Although necessary for proper growth and development, dysregulation of apoptosis has been associated with a variety of diseases including cancer and neurodegenerative disorders. Increased oxidative stress has also been associated with these diseases and has been shown to lead to cell death. Cell death can occur through a single pathway, or in concert involving multiple pathways. The goal of this study was to utilize multi‐parametric fluorescence microscopy to examine temporal characteristics of cell death after induction by various agonists. Using a fluorogenic caspase substrate in combination with a probe for oxidative stress, we observed that increased oxidative stress was followed by caspase activation after induction by several agonists. By simultaneously examining multiple parameters over time we were able to define the temporal progression of apoptosis relative to the onset of oxidative stress. Moreover, we were able to further characterize the mechanism of cell death by discriminating between cells which were apoptotic (active caspase‐3/7), autophagic (LC3B‐postive autophagosomes), or both. This multi‐parametric approach provided detailed information at the cellular level so that correlations and temporal resolution could be determined between oxidative stress and cell death mechanisms.