Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Partha Mukhopadhyay, Mohanraj Rajesh, G. Haskó, Brian J. Hawkins, Muniswamy Madesh, Pál Pacher

Research output: Contribution to journalArticle

249 Citations (Scopus)

Abstract

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes 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 production and execution of programmed cell death in virtually any cell type.

Original languageEnglish
Pages (from-to)2295-2301
Number of pages7
JournalNature Protocols
Volume2
Issue number9
DOIs
Publication statusPublished - Sep 2007

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Flow cytometry
Confocal microscopy
Confocal Microscopy
Superoxides
Flow Cytometry
Apoptosis
Annexin A5
Microscopy
Microscopic examination
Mitochondria
Endothelial cells
Cell death
Cell Death
Endothelial Cells
Cell Line

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. / Mukhopadhyay, Partha; Rajesh, Mohanraj; Haskó, G.; Hawkins, Brian J.; Madesh, Muniswamy; Pacher, Pál.

In: Nature Protocols, Vol. 2, No. 9, 09.2007, p. 2295-2301.

Research output: Contribution to journalArticle

Mukhopadhyay, Partha ; Rajesh, Mohanraj ; Haskó, G. ; Hawkins, Brian J. ; Madesh, Muniswamy ; Pacher, Pál. / Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. In: Nature Protocols. 2007 ; Vol. 2, No. 9. pp. 2295-2301.
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