High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria

George W. Rogers, Martin D. Brand, Susanna Petrosyan, Deepthi Ashok, Alvaro A. Elorza, David A. Ferrick, Anne N. Murphy

Resultado de la investigación: Article

235 Citas (Scopus)

Resumen

Recently developed technologies have enabled multi-well measurement of O 2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 μg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.

Idioma originalEnglish
Número de artículoe21746
PublicaciónPLoS ONE
Volumen6
N.º7
DOI
EstadoPublished - 2011

Huella dactilar

Heart Mitochondria
Mitochondria
Respiration
mitochondria
Throughput
Polarography
Smegmamorpha
Technology
Preclinical Drug Evaluations
Liver Mitochondrion
Mitochondrial Proteins
Respiratory Rate
Assays
Phosphoric Monoester Hydrolases
Protons
Liver
breathing
assays
Injections
heart

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Citar esto

Rogers, G. W., Brand, M. D., Petrosyan, S., Ashok, D., Elorza, A. A., Ferrick, D. A., & Murphy, A. N. (2011). High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS ONE, 6(7), [e21746]. https://doi.org/10.1371/journal.pone.0021746
Rogers, George W. ; Brand, Martin D. ; Petrosyan, Susanna ; Ashok, Deepthi ; Elorza, Alvaro A. ; Ferrick, David A. ; Murphy, Anne N. / High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. En: PLoS ONE. 2011 ; Vol. 6, N.º 7.
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High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. / Rogers, George W.; Brand, Martin D.; Petrosyan, Susanna; Ashok, Deepthi; Elorza, Alvaro A.; Ferrick, David A.; Murphy, Anne N.

En: PLoS ONE, Vol. 6, N.º 7, e21746, 2011.

Resultado de la investigación: Article

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