title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy.
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.
authors.affiliation: Section on Oxidative Stress Tissue Injury, Laboratories of Physiological Studies, National Institutes of Health/NIAAA, Bethesda, Maryland 20892, USA.
authors.forename: Partha
authors.initials: P
authors.lastname: Mukhopadhyay
authors.forename: Mohanraj
authors.initials: M
authors.lastname: Rajesh
authors.forename: György
authors.initials: G
authors.lastname: Haskó
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Hawkins
authors.forename: Muniswamy
authors.initials: M
authors.lastname: Madesh
authors.forename: Pál
authors.initials: P
authors.lastname: Pacher
mesh_terms: D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D009930:Organic Chemicals / Q000032:analysis; D010617:Phenanthridines / Q000032:analysis; D013481:Superoxides / Q000378:metabolism*
chemical_list: C521281:5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine; D017304:Annexin A5; D015415:Biomarkers; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D010617:Phenanthridines; C402795:SYTOX Green; D013481:Superoxides