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.
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Acknowledgements
We thank Ms. Sufen Zheng for her technical help for the studies.
Funding
This research project was supported by the National Institutes of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (NS094834—P.V. Katakam), National Institute on Aging (R01AG047296—R. Mostany), and National Institute of Diabetes and Digestive and Kidney Diseases (DK107694—R. Satou). In addition, the study was supported by American Heart Association (National Center Scientist Development Grant, 14SDG20490359—P.V. Katakam; Greatersoutheast Affiliate Predoctoral Fellowship Grant, 16PRE27790122—V.N. Sure; and Scientist Development Grant, 17SDG33410366—I. Rutkai), Louisiana Clinical and Translational Science Center (supported in part by U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the LACaTS to I. Rutkai), and Louisiana Board of Regents grants (RCS, LEQSF(2016-19)-RD-A-24—R. Mostany). This work was supported in part by [U54 GM104940] from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center (to I. Rutkai). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Sperling, J.A., Sakamuri, S.S.V.P., Albuck, A.L. et al. Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies. Neuromol Med 21, 493–504 (2019). https://doi.org/10.1007/s12017-019-08552-8
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DOI: https://doi.org/10.1007/s12017-019-08552-8