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Titolo:
Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry
Autore:
Pham, NA; Robinson, BH; Hedley, DW;
Indirizzi:
Princess Margaret Hosp, Ontario Canc Inst, Dept Med Oncol & Hematol, Toronto, ON M5G 2M9, Canada Princess Margaret Hosp Toronto ON Canada M5G 2M9 onto, ON M5G 2M9, Canada Princess Margaret Hosp, Ontario Canc Inst, Div Expt Therapeut, Toronto, ONM4X 1K9, Canada Princess Margaret Hosp Toronto ON Canada M4X 1K9 ronto, ONM4X 1K9, Canada Hosp Sick Children, Program Metab, Toronto, ON M5G 1X8, Canada Hosp Sick Children Toronto ON Canada M5G 1X8 Toronto, ON M5G 1X8, Canada
Titolo Testata:
CYTOMETRY
fascicolo: 4, volume: 41, anno: 2000,
pagine: 245 - 251
SICI:
0196-4763(200012)41:4<245:SDOMRC>2.0.ZU;2-Q
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELECTRON-TRANSPORT CHAIN; OXIDATIVE STRESS; COMPLEX-I; APOPTOSIS; INHIBITION; GLUTATHIONE; DYSFUNCTION; TRANSITION; GENERATION; MECHANISM;
Keywords:
confocal microscopy; mitochondria; respiration; ATP; respiratory chain; mitochondrial membrane potential; reactive oxygen intermediates; digitonin;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
24
Recensione:
Indirizzi per estratti:
Indirizzo: Hedley, DW Princess Margaret Hosp, Ontario Canc Inst, Dept Med Oncol & Hematol, 610 Univ Ave, Toronto, ON M5G 2M9, Canada Princess Margaret Hosp 610 Univ Ave Toronto ON Canada M5G 2M9 a
Citazione:
N.A. Pham et al., "Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry", CYTOMETRY, 41(4), 2000, pp. 245-251

Abstract

Background: Increased mitochondrial generation of reactive oxygen intermediates (ROT) due to defective respiratory chain activity has been implicatedin physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a now cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin. Methods: Flow cytometry was used to measure mitochondrial membrane potential (Delta Psi (m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy. Results: Activity of the respiratory chain complexes assessed by Delta Psi(m), responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the now cytometry method allows the simultaneous assessment of mitochondrial ROI generation. Thiswas confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy. Conclusions: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation. Cytometry 41.245-251, 2000. (C) 2000 Wiley-Liss, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 19/01/20 alle ore 20:24:01