Premium
Simultaneous quantitative detection of mitochondrial superoxide production and apoptosis in live cells.
Author(s) -
Mukhopadhyay Partha,
Rajesh Mohanraj,
Yoshihiro Kashiwaya,
Hawkins Brian J,
Madesh Muniswamy,
Pacher Pal
Publication year - 2008
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.22.1_supplement.1153.1
Subject(s) - superoxide , microbiology and biotechnology , mitochondrion , flow cytometry , programmed cell death , intracellular , dichlorofluorescein , apoptosis , annexin , organelle , biology , chemistry , biochemistry , enzyme
Experiments with isolated mitochondria have established that these organelles are pivotal intracellular sources of superoxide in a variety of pathophysiological conditions. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells. Here we show approximately 3–7 fold dose‐ and time‐dependent increase in mitochondrial superoxide production (measured by MitoSOX using flow cytometry and confocal microscopy) in rat cardiac derived H9c2 myocytes and/or in human coronary artery endothelial cells triggered by Antimycin A, Paraquat, Doxorubicin or high glucose. We also describe simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell‐based techniques are the tremendous speed (1–2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial‐temporal changes in mitochondrial superoxide generation and execution of programmed cell death in virtually any cell type.