Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies

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 fun...

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Bibliographic Details
Published in:Neuromolecular medicine 2019-12, Vol.21 (4), p.493-504
Main Authors: Sperling, Jared A., Sakamuri, Siva S. V. P., Albuck, Aaron L., Sure, Venkata N., Evans, Wesley R., Peterson, Nicholas R., Rutkai, Ibolya, Mostany, Ricardo, Satou, Ryousuke, Katakam, Prasad V. G.
Format: Article
Language:English
Subjects:
Adenosine Diphosphate - pharmacology
Animals
Biomedical and Life Sciences
Biomedicine
Brain - metabolism
Brain - ultrastructure
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology
Central nervous system
Electron transport chain
Electron Transport Complex I - metabolism
Electron Transport Complex II - metabolism
Fluorometry - instrumentation
Fluorometry - methods
High-Throughput Screening Assays - instrumentation
High-Throughput Screening Assays - methods
Hydrogen-Ion Concentration
Internal Medicine
Male
Mice
Mice, Inbred C57BL
Microchemistry - instrumentation
Microchemistry - methods
Mitochondria
Mitochondria - drug effects
Mitochondria - metabolism
Mitochondrial Membranes - drug effects
Mitochondrial Membranes - metabolism
Mitochondrial Proteins - analysis
Mitochondrial Proton-Translocating ATPases - antagonists & inhibitors
Neurology
Neurosciences
Oligomycin
Oligomycins - pharmacology
Original Paper
Oxidative Phosphorylation
Oximetry - instrumentation
Oximetry - methods
Oxygen - analysis
Oxygen Consumption - drug effects
Protons
Respiration
Stroke
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Summary: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.
ISSN:1535-1084
1559-1174
DOI:10.1007/s12017-019-08552-8