Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies.
Neuromolecular Med
; 21(4): 493-504, 2019 12.
Article
em En
| MEDLINE
| ID: mdl-31172441
ABSTRACT
Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Consumo de Oxigênio
/
Encéfalo
/
Oximetria
/
Ensaios de Triagem em Larga Escala
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Fluorometria
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Microquímica
/
Mitocôndrias
Limite:
Animals
Idioma:
En
Revista:
Neuromolecular Med
Assunto da revista:
BIOLOGIA MOLECULAR
/
NEUROLOGIA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Estados Unidos