Abstract
Measuring respiration rate can be a powerful way to assess energetic function in isolated mitochondria. Current, plate-based methods have several advantages over older, suspension-based systems, including greater throughput and the requirement of only μg quantities of material. In this chapter, we describe a plate-based method for measuring oxygen consumption by isolated adherent mitochondria.
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References
Warburg O (1923) Versuche an überlebendem Carcinom-Gewebe (Methoden). Biochem Zeitschr 142:317–333
Warburg O (1924) Verbesserte Methode zur Messung der Atmung und Glykolyse. Biochem Z 152:51–63
Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA (2006) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 292(1):C125–C136. https://doi.org/10.1152/ajpcell.00247.2006
Rogers GW, Brand MD, Petrosyan S, Ashok D, Elorza AA, Ferrick DA, Murphy AN (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.t001
Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435(2):297–312. https://doi.org/10.1113/expphysiol.2006.034330
Gerencser AA, Neilson A, Choi SW, Edman U, Yadava N, Oh RJ, Ferrick DA, Nicholls DG, Brand MD (2009) Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal Chem 81(16):6868–6878. https://doi.org/10.1021/ac900881z
Ashour B, Hansford RG (1983) Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria. Biochem J 214:725–736
Letellier T, Malgat M, Mazat JP (1993) Control of oxidative phosphorylation in rat muscle mitochondria: implications for mitochondrial myopathies. Biochim Biophys Acta 1141:58–64
Orr AL, Ashok D, Sarantos MR, Ng R, Shi T, Gerencser AA, Hughes RE, Brand MD (2014) Novel inhibitors of mitochondrial sn-glycerol 3-phosphate dehydrogenase. PLoS One 9(2):e89938. https://doi.org/10.1371/journal.pone.0089938
Orr AL, Ashok D, Sarantos MR, Shi T, Hughes RE, Brand MD (2013) Inhibitors of ROS production by the ubiquinone-binding site of mitochondrial complex I identified by chemical screening. Free Radic Biol Med 65:1047–1059. https://doi.org/10.1016/j.freeradbiomed.2013.08.170
Orr AL, Vargas L, Turk CN, Baaten JE, Matzen JT, Dardov VJ, Attle SJ, Li J, Quackenbush DC, Goncalves RL, Perevoshchikova IV, Petrassi HM, Meeusen SL, Ainscow EK, Brand MD (2015) Suppressors of superoxide production from mitochondrial complex III. Nat Chem Biol 11(11):834–836. https://doi.org/10.1038/nchembio.1910
Hey-Mogensen M, Goncalves RL, Orr AL, Brand MD (2014) Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria. Free Radic Biol Med 72:149–155. https://doi.org/10.1016/j.freeradbiomed.2014.04.007
Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P (2009) Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc 4(11):1582–1590. https://doi.org/10.1038/nprot.2009.151
Siess EA (1983) Influence of isolation media on the preservation of mitochondrial functions. Hoppe Seylers Z Physiol Chem 364:279–289
Whipps DE, Halestrap AP (1984) Rat liver mitochondria prepared in mannitol demonstrate increased mitochondrial volumes compared with mitochondria prepared in sucrose media. Biochem J 221:147–152
Corcelli A, Saponetti MS, Zaccagnino P, Lopalco P, Mastrodonato M, Liquori GE, Lorusso M (2010) Mitochondria isolated in nearly isotonic KCl buffer: focus on cardiolipin and organelle morphology. Biochim Biophys Acta 1798(3):681–687. https://doi.org/10.1016/j.bbamem.2010.01.005
Mildvan AS (1987) Role of magnesium and other divalent cations in ATP-utilizing enzymes. Magnesium 6(1):28–33
Panov AV, Vavilin VA, Lyakhovich VV, Brooks BR, Bonkovsky HL (2010) Effect of bovine serum albumin on mitochondrial respiration in the brain and liver of mice and rats. Bull Exp Biol Med 149:187–190
Mookerjee SA, Gerencser AA, Nicholls DG, Brand MD (2017) Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. J Biol Chem 292(17):7189–7207. https://doi.org/10.1074/jbc.M116.774471
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Mookerjee, S.A., Quinlan, C.L., Wong, HS., Dighe, P., Brand, M.D. (2018). Plate-Based Measurement of Respiration by Isolated Mitochondria. In: Palmeira, C., Moreno, A. (eds) Mitochondrial Bioenergetics. Methods in Molecular Biology, vol 1782. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7831-1_17
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DOI: https://doi.org/10.1007/978-1-4939-7831-1_17
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